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Extraction optimization, physicochemical characterization, and antioxidant activity of polysaccharides from Rhodosorus sp. SCSIO-45730

Microalgal polysaccharides have been reported in many studies due to their uniqueness, biocompatibility, and high value, and Rhodosorus sp. SCSIO-45730 was an excellent source of polysaccharides and β-glucans. However, the polysaccharides from the red unicellular alga Rhodosorus sp. SCSIO-45730 have barely been studied. In this work, hot water extraction of Rhodosorus sp. SCSIO-45730 polysaccharides (RSP) was optimized using response surface methodology (RSM) based on Box–Behnken design (BBD). The maximum RSP yield (9.29%) was achieved under the optimum extraction conditions: liquid–solid ratio of 50.00 mL g−1; extraction temperature of 84 °C; extraction time of 2 h; and extraction times of 5 times. The results of physicochemical characterization showed that RSP had high sulfate and uronic acid with content of 19.58% and 11.57%, respectively, rough layered structure, and mainly contained glucose, galactose, xylose, and galacturonic acid with mass percentages of 34.08%, 28.70%, 12.46%, and 12.10%. Furthermore, four kinds of antioxidant assays were carried out, and the results indicated that RSP had strong scavenging activities on ABTS and hydroxyl radical and moderate scavenging activities on DPPH and ferrous chelating ability. These results indicated that RSP showed potential as a promising source of antioxidants applied in food, pharmaceutical, and cosmetics industry. Supplementary Information The online version contains supplementary material available at 10.1007/s10811-021-02646-2.


Introduction
Oceans, with total planetary coverage of 70%, possess high biodiversity and are habitats for a large number of seaweeds and microalgae (Gaignard et al. 2019). Marine microalgae can produce easily modified, biocompatible, stable biodegradable, non-toxic, and highly safe biological-active compounds, such as polysaccharides, carotenoids, proteins, peptides, essential fatty acids, vitamins, and mineral oxides; hence, it has been identified as an expected supplement in the fields of biochemistry, biomass energy resources, and pharmacology (Raposo et al. 2013;Zhang et al. 2019). In particular, some unique polysaccharides exclusively present in rhodophytes and numerous bioactive properties such as anti-tumor, antidiabetic, antioxidant, antiviral, anticoagulant, and immunomodulatory effects of polysaccharides have been reported (Raposo et al. 2014;Fleita et al. 2015). Rhodosorus belongs to the division Rhodophyta or red algae and studies have mainly focused on the extraction and purification of phycoerythrin from the algal cells. The existing research shows that Rhodosorus sp. SCSIO-45730 is rich in polysaccharides and its content was maximized up to 242.6 mg L −1 day −1 (Dai et al. 2020), which is much higher than the polysaccharide productivity of Porphyridium cruentum (Sun et al. 2008). In addition, Rhodosorus sp. SCSIO-45730 with high biomass was considered as an optimal feedstock for the production of bioethanol and β-glucans (Dai et al. 2020).
However, there are still no studies focusing on the extraction, physicochemical characterization, and antioxidant activity of Rhodosorus sp. .
The selection of a suitable extraction method is a crucial step in the extraction of polysaccharides from raw materials, because this decision may affect the yield, composition, structure, and integrity of bioactive polysaccharides (Alboofetileh et al. 2019a). There are various methods available for the extraction of polysaccharides, including hot water, dilute acid, dilute alkali, microwave, ultrasonic, and enzyme extractions (Ardiles et al. 2020). Generally, hot water extraction is the most common method for extracting polysaccharides with the advantages of low cost, simple operation, no special equipment required, excellent quality of final product, and most suitable for preparation of neutral and acidic polysaccharides (Dong et al. 2016). In addition, hot water extraction is an important technology from an industrial point of view. The yield of this method largely depends on a liquid-to-solid ratio, extraction time, extraction temperature, and extraction times (Li et al. 2019b). Yu used response surface methodology (RSM) to optimize the extraction process of Angelica sinensis polysaccharides (ASP), and the water-solid ratio and the extraction time were found to be the most important factors (Yu et al. 2013). The optimal procedure is shown as follows: water-solid ratio was 5 mL g −1 , extraction time was 130 min, and extraction number was 5. According to Ai et al. (2013), the extraction process of ASP was optimized by an orthogonal experimental design, and the extraction yield of ASP was 5.6% under the optical extraction condition (water-solid ratio 6 mL g −1 , extraction time 180 min, extraction temperature 100 °C, and extraction times 4). Therefore, it is very important to optimize the extraction conditions of RSP for its future research, development and industrial application. RSM is a set of statistical and computational techniques that are composed of experiment designing, model building, evaluation of factors, and search for optimal conditional factors (Golbargi et al. 2021). RSM is often used to determine the influence of each factor and its interaction on the extraction rate and identify the optimal conditions statistically. Furthermore, this modelbased approach not only reduces the number of experiments but also greatly reduces development time and overall cost.
Polysaccharides have gained much research interest in health food, new drugs, and cosmetics, as gelling agents, thickeners, emulsifiers, and stabilizers that have been attributed to their chemical structures and biological activities. It has been proved that sulfated polysaccharides have shown promising inhibitory effects on preventing recent coronavirus disease 2019 (COVID-19) (Hans et al. 2020;Pereira and Critchley 2020). In addition, the antioxidant activity is one of the most studied aspects of polysaccharides. Natural polysaccharides isolated from Porphyra/Pyropia, Eucheuma, Kappaphycus, and Gracilaria have strong free radical scavenging activities and unique properties to prevent oxidative damage in living organisms (Aziz et al. 2020). Based on the literature reports, biological activities of polysaccharides vary with their physical-chemical structural features such as physical appearance, the ratio of constituent monosaccharides, and degree of sulfation (Venkatesan et al. 2019). For example, Chlorella pyrenoidosa polysaccharides (CPP) using 60%, 70%, and 85% final ethanol concentrations for precipitation were called CPP60, CPP70, and CPP85, respectively. All of them possessed antioxidant activity in vitro, but CPP70 exhibited stronger scavenging activity against superoxide, DPPH, and hydroxyl radicals compared with CPP60 and CPP85, which resulted from the differences of structural features and molecular size of polysaccharides . Similarly, Laminaria polysaccharide with higher uronic acid and sulfate content displayed stronger antioxidant activity (Bhadja et al. 2016). Yuan and Macquarrie (2015) also reported that the scavenging capacity to DPPH radical of polysaccharides extracted from Ascophyllum nodosum is proportional to their -SO 3 H content, i.e., the component with 28.60% -SO 3 H content has the highest scavenging capacity of about 15%, whereas the component with 7.82% -SO 3 H content only has a scavenging capacity of about 2%.
As far as we know, there have been no reports on Rhodosorus sp. SCSIO-45730 polysaccharides (RSP). Therefore, this is a systematic study to optimize the condition for maximizing the hot water extraction yield of RSP using RSM. The structural and morphological features of RSP were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD). Besides, the antioxidant capacity of RSP was investigated via 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis 3-ethylbenzthiazoline-6-sulphonic acid (ABTS), hydroxyl radical scavenging, and ferrous chelating ability. These results provide a potential of polysaccharide from Rhodosorus sp. SCSIO-45730 in the food, pharmaceutical and cosmetics industry as an antioxidant.

Cultivation and collection of microalgae
Rhodosorus sp. SCSIO-45730 was isolated from Xisha Islands, South China Sea (111° 45.000′ E, 16° 28.471′ N). The species was identified according to the distinguishing morphological features, 18S rRNA gene sequences, and BLAST analysis reported previously (Dai et al. 2020). The strain was cultured in 1500-mL vertical bubble column photobioreactors (6.0 cm × 60 cm) containing seawater medium composed of 17.6 mM NaNO 3 ; 0.69 mM  (Li et al. 2019a). The cultures were incubated at 25 °C under continuous illumination with the light intensity gradually increased from 30 to 180 μmol photons m −2 s −1 in the first 4 days of cultivation and then kept at 180 μmol photons m −2 s −1 . The cells were harvested by centrifugation at 3214 × g for 5 min under the later stage of the exponential/linear phase and then washed twice with sterile deionized water, freezedried, ground into powder and stored before extraction (Fleita et al. 2015).

Hot water extraction of polysaccharides
RSP were extracted by a slight modification of the hotwater extraction method described by Khan et al (2019). Briefly, deionized distilled water was added to Rhodosorus sp. SCSIO-45730, and the resultant solutions were kept at room temperature for 30 min before heat treatment in a water bath. A preliminary single-factor experiment was carried out at a liquid-solid ratio (20-60 mL g −1 ), extraction temperature (60-100 °C), extraction time (1-5 h), and extraction times (2-6 times), respectively. When measuring the effects of the liquid-solid ratio (20,30,40,50, and 60 mL g −1 ) on the yield of the RSP, the extraction temperature was fixed at 90 °C, the extraction time was set to 2 h, and the extraction times was fixed at 3 times. When the effects of extraction temperature (60, 70, 80, 90, and 100 °C) on the RSP yield were determined, the liquid-solid ratio was set as 30 mL g −1 , the extraction time was fixed at 2 h, and the extraction times was set to 3 times. When estimating the effects of extraction time (1, 2, 3, 4, and 5 h) on the yield of the RSP, the liquid-solid ratio, the extraction temperature, and the extraction times were fixed at 30 mL g −1 , 90 °C, and 3 times, respectively. When assaying the effects of the extraction times (2, 3, 4, 5, and 6 times) on the yield of the RSP, the liquid-solid ratio, the extraction temperature and the extraction time were fixed at 30 mL g −1 , 90 °C, and 2 h, respectively. After performing the extraction process, the crude extract was centrifuged at 8228 × g for 10 min, and the supernatant was collected. A rotary evaporator was employed to concentrate the filtrate equal to 1/4 of its original volume at 50 °C, followed by precipitation through the addition of threefold ethanol (95%) and then stored at 4 °C overnight. Twenty milliliter of 20% hydrogen peroxide was added to the precipitate collected by centrifugation (12,857 × g, 10 min), which were stirred in a water bath at 50 °C for 1 h, and then heated to remove hydrogen peroxide completely. The supernatant was centrifuged at 12,857 × g for 10 min, dialyzed for 24 h (500 Da cutoff), and subsequently subjected to freeze-drying for 48 h to obtain RSP powder. Extraction yield (Y) was calculated by the following Eq. (1) ):

Experimental design and response surface modeling
According to the single-factor experiment results (Fig. S1), the Box-Behnken design (BBD) in the present study was a design of four variables: liquid-solid ratio (A) at 30, 40, and 50 mL g −1 ; extraction temperature (B) at 70, 80, and 90 °C; extraction time (C) at 1, 2, and 3 h; and extraction times (D) at 3, 4, and 5 times. Each variable had three different levels (− 1, 0, 1). The extraction yield of RSP was used as the dependent variable. The whole design contains 27 experimental runs, which were represented by the coded and non-coded values of the experimental variables as shown in Table 1. The experiment was carried out in random order. The mutual interaction among the variables and their corresponding optimum levels were expressed by a secondorder polynomial equation (Awatief et al. 2016). Furthermore, three additional confirmation extraction experiments under the optimal conditions were carried out to verify the accuracy of the statistical experimental strategies (Shang et al. 2021).

Chemical composition analysis
The content of total sugar was determined according to the method of phenol-sulfuric acid using glucose as a standard . Bicinchonininc acid (BCA) protein colorimetry was used to measure the protein content of RSP . The sulfate content of RSP was analyzed by the BaCl 2 -gelatin turbidity method of Bhadja et al (2016) with some modifications. Briefly, about 10 mg of RSP sample and K 2 SO 4 standard were solubilized in 5 mL of 4 M HCl, hydrolyzed at 90 °C for 2.5 h. The hydrolyzed samples (0.1 mL) were pipetted into 5-mL centrifuge tubes, to which 1.9 mL of 3% TCA and 0.5 mL of BaCl 2gelatin reagent were added. The released barium sulfate suspension was measured at λ = 360 nm. Based on a K 2 SO 4 calibration curve (0.52-200 mg mL −1 ), the sulfate content of RSP was quantified. The uronic acid content of RSP was measured by the meta-hydroxydiphenyl assay method with modification (Hui et al. 2019;Hao et al. 2020). RSP were dissolved in water at a concentration of 0.1 mg mL −1 , and different concentrations (0, 0.02, 0.03, 0.04, 0.05, 0.06, and 0.08 mg mL −1 ) of the standard, galacturonic acid (GalA) were also prepared. A total of 0.5 mL of sample and standard were mixed with 3 mL of sodium tetraborate sulfuric acid solution (0.0125 mol L −1 ), heated at 90 °C for 15 min, and then cooled down in an ice bath to terminate the reaction. Finally, 50 uL of meta-hydroxydiphenyl solution (0.15%) was accurately added and mixed and kept at room temperature for 40 min. The absorbance was measured at 520 nm. The contents of uronic acids in samples could be calculated according to the standard curve. Total phenolic content (TPC) of RSP was determined using the Folin-Ciocalteu reagent assay (Foo et al. 2017). Based on a gallic acid calibration curve, the TPC was determined and indicated as mg gallic acid equivalent (GAE) per g dry biomass (Almendinger et al. 2021).

Monosaccharide composition
Ion chromatography (Thermo Fisher Scientific, USA) was applied to analyze the monosaccharide composition of RSP with 13 standard monosaccharides (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid). Five mg of polysaccharide sample was accurately weighed, and 1 mL TFA acid (2.5 M) solution was added and heated at 121 °C for 2 h, followed by drying under nitrogen. Methanol was added to clean it and then dried under nitrogen. The methanol cleaning step was repeated 2-3 times. The sample was then dissolved in sterile water and transferred to the chromatographic flask for testing. An electrochemical detector was employed to detect the monosaccharide components. Mobile phase A was 0.1 M NaOH, and the mobile phase B was 0.1 M NaOH and 0.2 M NaAC. With 5 μL of injection volume, the samples were analyzed on a Dionex CarboPac PA10 column (250 × 4.0 mm, 10 um) at 30 °C with a flow rate of 0.5 mL min −1 .
SEM The surface characterization and microstructure evaluation of RSP were observed by SEM (Sigma 300/VP, Zeiss) at a voltage of 3.0 kV and under 10,000 × and 50,000 × mag- nification, respectively. Freeze-dried RSP samples were attached to specimen holders with double-sided carbon tape and then sputtered with gold for analysis under a high-vacuum chamber .
AFM A scanning probe microscope (Nano Man VS) was used for AFM analysis of RSP. The RSP sample was placed in distilled water (1 µg mL −1 ) and sonicated for 30 min. After sonication, 2-3 µL of RSP solution was pipetted onto mica disc which was subsequently dried at 120 °C for 0.5 min before analysis .
XRD XRD analyses were carried out at room temperature with Cu Kα radiation on a MXP18 HF diffractometer (MAC Science Co., Japan). The patterns were collected between 10 and 90° at a scan rate of 10.0° min −1 .
FT-IR FT-IR (IR Affinity-1, Japan) was used to analyze the functional chemistry of RSP. Infrared spectroscopy of the sample was recorded at the frequency of 400-4000 cm −1 at a resolution ratio of 1 cm −1 , and the scan number was 32 (Guidara et al. 2021).

Antioxidant activities of RSP
DPPH radical scavenging activity assay The free-radical scavenging capacity of RSP was analyzed using the DPPH test based on Venkatesan et al. (2019). Briefly, 100 uL of 0.1 mM DPPH methanolic solution was added to the 100 uL RSP of different concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 mg mL −1 ). The solution was vortexed for 1 min and kept at room temperature for 30 min in the dark, and the absorbance at 517 nm was measured by a micro-plate reader. Ascorbic acid was used as reference standard. The ability to scavenge the DPPH radical was calculated as follows: Here, DPPH solution plus RSP sample was used as A sample , RSP sample without DPPH solution was used as A sample blank , and DPPH solution without RSP sample was used as A control .
ABTS radical scavenging activity assay ABTS radical scavenging activity was measured by the method of Liu et al. (2015). ABTS radical cation solution was prepared by 12-16 h reaction of ABTS (7.4 mM) with potassium persulfate (2.6 mM) at room temperature in dark. The solution was diluted with H 2 O by 20.3 times to obtain an absorbance of 0.700 at 734 nm. 0.2 mL samples of different concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 mg mL −1 ) were mixed with the diluted ABTS (0.8 mL). The mixture was shaken for 10 s and let stand for 6 min. The absorbance at 734 nm was measured with a micro-plate reader. Ascorbic acid was used as a positive control. The activity to scavenge ABTS radical was calculated with the formula below: where A sample contains ABTS solution and RSP solution, A sample blank contains RSP solution and H 2 O, and A control contains ABTS solution and H 2 O.
Hydroxyl radical scavenging activity assay The hydroxyl radical scavenging activity assay was done as previously described with some modifications . A total of 1.0 mL of 9.0 mM FeSO 4 and 1.0 mL of 9.0 mM ethanol salicylate were added to 1.0 mL of polysaccharide solution in 5-mL test tubes, and 1.0 mL of 9.0 mM H 2 O 2 was added. The reaction was carried out in a 37 °C water bath for 30 min, and the absorbance was measured at 510 nm. Each polysaccharide sample and the positive control (V c ) had five concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 mg mL −1 ). The hydroxyl radical scavenging activity was calculated as follows: where A sample is the absorbance of the RSP sample, A sample blank is the absorbance of the reagent blank (water instead of H 2 O 2 ), and A control is the absorbance of the blank without the RSP sample.
Ferrous ion chelating activity assay The iron chelating properties of RSP were determined according to Alencar et al. (2019). Briefly, 0.05 mL of 2 mM FeCl 2 , 0.5 mL sample of RSP at different concentrations (0.125, 0.25, 0.5, 1.0, and 2.0 mg mL −1 ), and 0.1 mL of 5 mM ferrozine were mixed. The mixtures were vortexed for 1 min and reacted 10 min before measuring at 562 nm. EDTA-2Na was used as standard. The chelating ability of ferrous ion was calculated by the following formula: where A sample stands for the absorbance of the reaction mixture, A sample blank stands for reagents without the RSP sample, and A control stands for the absorbance of the RSP sample without reagents. (3)

Statistical analysis
Statistical analysis was carried out using Design-Expert Software (8.0.6) and IBM SPSS statistics 25 (SPSS Inc., USA) software. One-way analysis of variance (ANOVA) was used. The polysaccharides extraction experimental results are expressed as mean and standard deviations of triplicate experiments (n = 3). The experimental data of chemical composition and antioxidant activities of RSP are shown as mean and standard deviations of two independent biological replicates and three technical replicates. P < 0.05 was considered statistically significant.

Model fitting and statistical analysis
As shown in Table 1, a 27-run BBD was applied to investigate the combined effects of four independent variables on polysaccharide yield of Rhodosorus sp. SCSIO-45730. The experimental results obtained based on BBD f it a second-order polynomial mathematical equation in the coded level as follows:

Effect of interacting variables
Three-dimensional response surface plots (Fig. 1) were established to illustrate the relationship between the independent variables and the response and the predicted optimal extraction parameters for maximizing the yield of RSP. The two-dimensional response surface plots (Fig. 2) also displayed modes of the regression equation in which an elliptical contour plot manifests the interactions are significant, and in turn, a circular contour plot means the interactions are negligible. As shown in Figs. 1 and 2, interaction effects between liquid-solid ratio (A) and extraction temperature (B), liquid-solid ratio (A) and extraction time (C), liquid-solid ratio (A) and extraction times (D), extraction temperature (B) and extraction time (C), extraction temperature (B) and extraction times (D), and extraction time (C) and extraction times (D) were observed visually. When extraction time and extraction times were fixed at the zero levels, the polysaccharide yield increased at the beginning but then decreased with increasing liquid-solid ratio and extraction temperature. The liquid-solid ratio and extraction temperature belonged to insignificant interactions (Figs. 1a and 2a). Figures 1b and 2b described the extraction yield was not changed significantly by increasing the extraction time, but it increased as the ratio of water-to-raw-material increased under the zero level of extraction temperature and extraction times. Figures 1c and 2c showed the polysaccharide yields increased with enhanced liquid-solid ratio and extraction times but without significant effects. In Fig. 1d and 2d, the results suggested that extraction temperature and extraction time exerted a notable impact on polysaccharide yield. On the contrary, Figs. 1e and 2e indicated that the influence of extraction temperature and extraction times was not significant when liquid-solid ratio and extraction time was fixed at zero levels. Similarly, the mutual interaction between extraction time and extraction times was not significant on polysaccharide yield when liquid-solid ratio and the extraction temperature was fixed to be level 0 ( Figs. 1f and 2f).

Optimization and model verification
The optimum extraction conditions were obtained by solving the regression equation using the Design-Expert Software, it was concluded that the optimal extraction conditions were as follows: liquid-solid ratio, 50 mL g −1 ; extraction temperature, 84.41 °C; extraction time, 2.04 h; and extraction times, 5 times. Under these optimal conditions, the maximum predicted extraction yield was 9.30%. Considering the operation in actual production, the adjusted optimum extraction conditions were as follows: liquid-solid ratio of 50.00 mL g −1 ; extraction temperature of 84 °C; extraction time of 2 h; and Polysaccharide yield(%) = 0.57 + 1.45A + 0.51B + 0.14C Table S1 shows the ANOVA results for the fitted quadratic polynomial model of extraction of RSP. The high F value (19.38) and the low P value (< 0.0001) of the model indicated the response surface quadratic model was significant. The values of the regression coefficient (R 2 ), the value for the adjusted coefficient of determination (R 2 adj) and coefficient of variation (CV) was 0.9576, 0.9082, and 9.81% respectively. "Adeq Precision" measures the signalto-noise ratio, and a ratio greater than 4 is desirable. The ratio of 17.521 indicates an adequate signal that means this model can be used to navigate the design space. The significance of each coefficient was checked using F value and P value. In the current study, it could be seen that the regression coefficients (A, D, A 2 , and C 2 ) exhibited a highly significant effect (P < 0.001) and the regression coefficients (B, BC, and B 2 ) were significant effects (P < 0.05). The other term coefficients were insignificant (P > 0.05). The normality assumption was illustrated by constructing a normal probability plot of the residuals. extraction times of 5 times. Triplicate confirmatory experiments under the modified optimal condition for the RSP extraction were carried out, and the average extraction yield was 9.29%, which confirmed the validation of the response model and the suitability of the optimal conditions for the extraction of Rhodosorus sp. SCSIO-45730.

Chemical composition and monosaccharide composition
The proximate compositions of RSP are summarized in Table 2. As shown, the content of the total sugar and protein in RSP was 49.62% and 6.16%, respectively. A sulfate content of 19.58% and a uronic acid content of 11.57% were attributed to the extracted RSP, respectively. The total phenolic content was 2.16%. The monosaccharide compositions of RSP were determined via ion chromatography analysis after hydrolysis using trifluoroacetic acid. RSP contained twelve kinds of monosaccharides in the form of glucose, galactose, xylose, galacturonic acid, mannose, ribose, fructose, mannuronic acid, rhamnose, fucose, arabinose, and guluronic acid, in which the retention time of the first four monosaccharides were 10.96 min, 9.46 min, 12.90 min, and 33.93 min (Fig. S2), respectively. RSP mainly contained glucose, galactose, xylose, and galacturonic acid with mass percentages of 34.08%, 28.70%, 12.46%, and 12.10% (Table 2), respectively.

Structural and morphological characterization
SEM is a considerable powerful tool to monitor the morphological properties of the extracted polysaccharides, for instance, porosity, size, and shape of macromolecules. As shown in Figs. 3a and 3b, RSP is a relatively uniform sheet structure with a rough appearance. The 3D topography, surface roughness, and height of RSP were expounded by AFM (Fig. 3 c, d, and e). Somewhat elongated particles with irregular shape and size pointing towards random coil conformation of RSP were displayed, wherein the vertical distance was 5.86 nm, the horizontal distance was 249 nm, and the variations in height yielded an average of 1.95 nm, with the shortest being 0.04 nm and the tallest 6.46 nm. Figure 4a shows the FTIR spectrum of RSP between 4000 and 500 cm −1 . Both the peaks at 3448 cm −1 and 2926 cm −1 were attributed to the stretching vibration of O-H and C-H, respectively, indicating the typical absorption peaks of polysaccharides. Both the signals at 1618 cm −1 and 1412 cm −1 were associated with the presence of carbonyl groups, indicating that GPOP-1 contained uronic acid (Cui et al. 2019). The band at 1218 cm −1 was due to the stretching vibration of O = S = O, indicating the presence of sulfate in the sample (Sun et al. 2014). The strong absorption at 1010 cm −1 was dominated by the stretching vibration of the pyranose ring, confirming that the extracted RSP contained pyranose sugar ). Moreover, a medium peak at 930 cm −1 and a weak peak at 866 cm −1 belonged to the beta and alpha configuration. Hence, β-glycosidic bonds were the main type followed by few α-glycosidic bonds in RSP. Figure 4b shows that the XRD pattern revealed a strong diffraction peak at 2θ = 20.88, manifesting that RSP were semi-crystalline polymers. Consequently, it confirmed that RSP had both crystalline and amorphous portions in their structure.

Antioxidant activities analysis
As shown in Fig. 5a, there was a dose-dependent effect observed between the concentrations of RSP and the DPPH scavenging effect. More specifically, the lowest DPPH scavenging effect (24.34%) was observed at 0.0625 mg mL −1 , while a concentration of 2 mg mL −1 yielded the highest clearance rate of 46.74% for DPPH. In the present work, the IC 50 values (concentration of the extract capable of scavenging 50% of DPPH) of RSP were 3.00 mg mL −1 (Table S2).
The ABTS radical scavenging abilities of RSP are shown in Fig. 5b. Similar to DPPH radical scavenging activity, the ABTS scavenging ability improved as the RSP concentration increased from 0.0625 to 2.0 mg mL −1 . The highest scavenging effect of RSP was 64.42% at a concentration of 2 mg mL −1 in comparison to 94.83% for vitamin C (P < 0.05). The IC 50 values of RSP were 1.74 mg mL −1 (Table S2). Figure 5c depicts the effects of scavenging hydroxyl radicals by RSP. The scavenging effects were in a concentration-dependent way, in which the scavenging rate varies greatly with the concentration in the range of 0.0625 to 0.125 mg mL −1 , while the scavenging percentage varies slightly with the concentration in the range of 0.125 to 2.00 mg mL −1 . RSP scavenged hydroxyl radical to a greater extent (65.32%) at the highest concentration of 2 mg mL −1 , and the lowest activities of hydroxyl radical scavenging were 4.57% at 0.125 mg mL −1 . The IC 50 value of the RSP was 0.83 mg mL −1 (Table S2).
The ferrous chelating ability of RSP at different concentrations is presented in Fig. 5d. It is obvious that the ferrous ion-chelating activity of RSP kept increasing with dose, with a rate of 43.19% at 2.0 mg mL −1 , while EDTA-2Na control showed a ferrous chelating ability of 99.05% at the same concentration (P < 0.05). The IC 50 value was 2.76 mg mL −1 for the RSP and 0.01 mg mL −1 for EDTA-2Na (Table S2).

Optimization of RSM for RSP extraction
In this study, the hot water extraction technology of RSP was established, and a mathematical model between the factors and the extraction rate of RSP was constructed. The performance of the fitting models was expressed as R 2 correlation, and significant analyses were performed using F-test . The values of R 2 , R 2 adj, and CV demonstrated that the model was remarkably significant, reasonable, reproducible, and reliable (Hu et al. 2020). In addition, the normality assumption was satisfied as to the residual plots approximated along a straight line and the residuals from the least squares fit to show the adequacy of the fitted RSM (Golbargi et al. 2021).
Based on the significance, reliability, and adequacy of the model, we concluded that the model can fully reflect the relationship between the efficiency of hot water extraction of RSP and the four factors including liquid-to solid ratio, extraction temperature, extraction time, and extraction times. With the increase of the liquid-to-solid ratio, extraction temperature, extraction time, and extraction times, the yield of RSP all showed a trend of increasing first and then decreasing. The higher liquid-solid ratio, extraction temperature, extraction time, and extraction times may cause softening of the algal tissue, increasing the solubility of the polysaccharides, which improves the rate of diffusion, thus giving a higher rate of extraction . In addition, the density and viscosity of the extract decreased with increasing extraction temperature, time, and times, which was beneficial to extracting buffer into the algal matrix. Nevertheless, the polysaccharide started to decompose when the extraction temperature, time, and times were too high. With the increase of liquid-solid ratio, the increased viscosity of the solvent caused an extension of diffusion distance for the microalgal cells leading to the slow increase of extraction of polysaccharides. Besides, excessive water would weaken the cavitation effect from the extraction process, thus reducing 12.10 ± 0.45 Glucuronic acid 0.84 ± 0.14 Mannuronic acid 0.14 ± 0.02 Glucuronic acid ND the extraction of polysaccharides. Cho et al. (2019) reported that the polysaccharides yield from Giant African snail using pressurized hot water extraction increased with an increase in the temperature from 100 to 150 °C, while the yield dropped dramatically to 2.53% at 250 °C. The polysaccharides yield from Giant African snail also increased first and then decreased with the increase of solid-liquid ratio (Cho et al. 2019). At present, there is no multi-factor optimization test for hot water extraction of RSP, and the response surface optimization method is used to achieve an extraction rate of 9.29%. It is worth mentioning here that the RSP yield in this study using the optimum extraction was markedly higher than that previously common hot water extraction with an extraction yield of 3.13% (Fig. S1). This result demonstrates the value of response surface optimization for the further development and improvement of RSP extraction technology. However, hot water extraction method is conventionally used, and it is not compared with any other methodology using modern extraction technology in this study. In the future, we plan to explore various advanced polysaccharide extraction techniques, such as microwave-assisted, ultrasonic-assisted, and enzyme-assisted extraction, promoting the large industrial preparation of RSP (Yuan et al. 2020a).

Physicochemical characteristics of RSP analysis
In general, the chemical structure of polysaccharides determines their physical, chemical, and biochemical properties as well as their biological activities (Maria et al. 2015). The total sugar content of RSP (49.62%) is appropriately consistent with the content that has been reported in Gracilaria chouae sulfated polysaccharides (Khan et al. 2019). Conversely, the carbohydrate content of Nizamuddinia zanardinii was 45.87% by ultrasoundmicrowave extraction method and 62.04% by cellulase extraction (Alboofetileh et al. 2019b). Research shows that the chemical composition of microalgal polysaccharides depends on algal species, population age, environmental conditions, geographic location, seaweed harvest season, the isolation, and purification methods used (Palanisamy et al. 2017). The protein in RSP existed mostly because they are part of the structure of cell walls and closely associated with polysaccharides (Yaich et al. 2014). The sulfate content of RSP (19.58%) is higher than that previously observed for the extracted polysaccharides from the red alga Porphyra haitanensis, which was almost 17.06% (Wang et al. 2013). Sun et al. (2014) reported that the uronic acids content of extracellular polysaccharides from Microcystic aeruginosa was 10.7% that lower than our reported content. It is known that for polysaccharides from natural products, the existence of sulfate and uronic acid in the molecular structure is responsible for key metabolic activity in living systems. Moreover, higher sulfate and uronic acid content induced higher antioxidant capacity, antiviral, and anticancer properties (Yaich et al. 2014). Wang et al. (2010) reported an increased antioxidant capacity of fucoidan with increasing sulfate and uronic acid content. It was also found that exopolysaccharides (EPS) extracts with a higher sulfate and uronic acid content revealed a strong activity against V. stomatitis virus (Raposo et al. 2014). In addition, Koyanagi et al (2003)  for the structural characterization and bioactive studies of polysaccharides and also helpful for the quality control of functional polysaccharides . The kind of monosaccharide composition in Rhodosorus sp. SCSIO-45730 commonly appears in red algae polysaccharides, as described in the review of Cheong et al (2018). The structural and morphological characterization of RSP not only is related to the function and applications of polysaccharides, but also favorable to understand the phylogeny of the species differences among various algae (Shimonaga et al. 2008). Similar SEM observations of uniform sheet structure with a rough appearance have been reported by Liu et al. for polysaccharides extracted from Sargassum fusiforme . Research also shows that the smoothness of the microstructure surface of the extracted polysaccharides can safely be related to the feature of sulfated polysaccharides electron micrographs . From AFM, it can be seen that the size of polysaccharide molecules is much larger than the theoretical value of single chain diameter (0.1-1.0 nm), indicating that the polysaccharide unit could be branched and tangled with each other, which may be due to van der Waals force interaction between polysaccharide molecules and hydrogen bond association between sugar chains (Ji et al. 2021).

In vitro antioxidant activities of RSP analysis
DPPH radical scavenging has been extensively used to evaluate the free radical scavenging activities of bioactive compounds. Previous literature reports have indicated that sulfated polysaccharides derived from Porphyra haitanensis exhibited a DPPH scavenging effect of 34.63% at 2 mg mL −1 ) and 3 mg mL −1 Gracilaria chouae polysaccharide showed 22.3% clearance rate (Khan et al. 2019). The IC 50 of polysaccharides from the green alga Ulva lactuca (62.13 mg mL −1 ) and the benthic diatom Nitzschia longissima (70.83 mg mL −1 ) were higher than those found in the present investigation (Kokabi et al. 2013;Gopinath and Sampathkumar 2014). The free radical scavenging activity of pineapple core polysaccharides may be mainly caused by the presence of hydroxyl and phosphoric acid in the structure (mainly galacturonic acid) (Hadidi et al. 2020). The polyhydroxy hydrogen donor can scavenge DPPH free radicals, thereby reducing the effect of oxidative stress.
The ABTS scavenging ability improved as the RSP concentration increased, which was in agreement with the electron transfer capability and antioxidant potency of polysaccharides. Khan et al. (2019) found that sulfated polysaccharides of G. chouae possessed an ABTS scavenging ability of 19.07% at 3 mg mL −1 . Based on these results, it can be preliminarily concluded that the extracted RSP has potential in acting as ABTS antioxidants.
Hydroxyl radical may be a by-product of immune action, so it has a short life span. These free radicals are highly reactive and can easily cross the cell membrane at specific positions, react with most biological macromolecules, such as carbohydrates, nucleic acids, lipids, and amino acids, resulting in human health damage. Therefore, it is very important to eliminate unnecessary hydroxyl radicals (Song et al. 2018). The polysaccharides of F1, F2, and F3 from L. japonica showed significant scavenging activities on hydroxyl radicals in a concentration-dependent manner, at the concentration of 5 mg mL −1 , reaching 35, 60, and 65%, respectively (Song et al. 2018). Xia et al. (2014) reported a polysaccharide extracted from the marine diatom Odontella aurita with a hydroxyl radical scavenging effect of approximately 42% at 2.0 mg mL −1 . The RSP had a significant effect on hydroxyl radical scavenging effects in this study might be due to sulfate content and uronic acid content, which is consistent with earlier studies ). The polysaccharide can show good radical scavenging effects in antioxidant tests only when sulfate content and uronic acid content are at a higher level.
Ferrous ions can generate reactive oxygen species by the Fenton free radical reaction, leading to cell oxidative damage . Henceforth, the chelating influence on ferrous ions has been lately broadly used to assess some antioxidant activity of polysaccharides (Huang et al. 2021). The iron-chelating ability of polysaccharides may be associated with the formation of cross-bridges between the carboxyl group in uronic acid and the divalent ions . The chelating rate of a novel heteropolysaccharide obtained from A. pubescens root was close to our present investigation at 2.0 mg mL −1 (Yuan et al. 2020b). On the whole, RSP was found moderately effective in chelating ferrous irons.
Based on the results of RSP on four different free radical-scavenging experiments, RSP has stronger in vitro antioxidant activity. The antioxidant activity of RSP is closely related to the main chain, main chain bond type (such as monosaccharide composition and sugar chain bond type), branched chain structure (such as substitution position and degree of substitution), and high-level configuration. From our study, the reason for the mechanisms in antioxidant activity of RSP cannot be drawn clearly. In the future, we will plan to deeply investigate structure-activity relationships and mechanisms of RSP.

Conclusion
This study is the first to report the extraction of RSP with its physicochemical characterization and antioxidant evaluation. RSM was used to optimize the extraction conditions, which effectively improved the extraction efficiency. The final optimization conditions were detailed as follows: liquid-solid ratio, 50.00 mL g −1 ; extraction temperature, 84 °C; extraction time, 2 h; and extraction times, 5 times. Under these optimal conditions, the maximum extraction yield was 9.29%. RSP was found to present a rough lamella structure with high sulfate content and uronic acid and mainly comprise glucose, galactose, xylose, and galacturonic acid. In addition, the antioxidant analyses disclosed the RSP exhibited strong hydroxyl radical scavenging ability (IC 50 = 0.83 mg mL −1 ), followed by ABTS radical scavenging ability (IC 50 = 1.74 mg mL −1 ), ferrous chelating ability (IC 50 = 2.76 mg mL −1 ), and DPPH radical scavenging ability (IC 50 = 3.00 mg mL −1 ). The present work provides a theoretical basis for the application of RSP as natural nontoxic antioxidants, potential functional food ingredients, and nutraceutical agents of biomedical. However, investigation on the structure-activity relationships and mechanism of polysaccharides is still required.