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H2O2 and its effects on structural characteristics
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Degradation of polysaccharides from Sargassum fusiforme using UV/H2O2 and its effects on structural characteristics Xiaoyong Chena, Ruifen Zhangb, Yizhou Lia,c, Xiong Lia,c, Lijun Youa,c,*, Viktoryia Kulikouskayad, Kseniya Hileuskayad a
School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong, 510640, China Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture and Rural Affairs/ Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou, Guangdong, 510610, China c Overseas Expertise Introduction Center for Food Nutrition and Human Health (111 Center), Guangzhou, Guangdong, 510640, China d Institute of Chemistry of New Materials, National Academy of Sciences of Belarus, Minsk, 220141, Belarus b
ARTICLE INFO
ABSTRACT
Keywords: UV H2O2 Polysaccharides Degradation Sargassum fusiforme
The depolymerization effect of UV/H2O2 on the polysaccharides from Sargassum fusiforme (PSF), a brown algae, were studied. The structural changes of PSF before and after UV/H2O2 treatment were analyzed, and molecular weight changes during in vitro digestion were determined. Results indicated that the molecular weight of PSF was reduced from ∼289 to ∼12.6 kDa within 2 h with UV/150 mmol/L H2O2, and the depolymerization effect of UV/H2O2 was significantly higher than that of UV or H2O2 alone. In addition, the UV/H2O2 treatment had a high recovery rate of total sugar (93.54 %) and clearance rate of protein (76.34 %). The monosaccharide composition showed that UV/H2O2 treatment could increase the mole percentage of mannose (37.44 %) and decrease the mole percentage of fucose (14.88 %). The helix-coil transition, X-ray diffraction (XRD) and atomic force microscopy (AFM) imaging showed that the UV/H2O2 treatment depolymerized PSF. Rheological studies indicated that PSF with UV/H2O2 treatment had lower viscosity. In vitro digestion showed that PSF was minimally digested with the in vitro gastrointestinal tract simulation, but PSF with UV/H2O2 treatment could be digested in the low acid environment in the simulated gastric juice, but was minimally digested with the simulated intestinal juice. This studied suggested that the preparation and application of functional PSF with low molecular weight might be beneficial.
1. Introduction The poor degradation of polysaccharides with high molecular weights (MW) is of concern because of poor solubility and bioavailability (Zhang, Wang, Zhao, & Qi, 2014). Various degradation methods have been proposed, including chemical, physical and biological methods. However, chemical methods (such as acid or alkali) have the advantage of simple operations, but may have serious safety and environmental problems (Ren et al., 2017; Xiao, Sun, & Sun, 2001). Physical methods (such as ultrasound, irradiation, microwave, high pressure microfluidization and pulsed electric field) can reduce environmental pollution, but require specialized equipment, and are difficult to do in an industrial environment (Zhou & Ma, 2006). Biological methods (such as enzymatic hydrolysis and microbial fermentation) can be done using mild reaction conditions, good safety, and low cost
features, but screening of enzymes and microbes that can degrade polysaccharides is a difficult problem due to the complicated structures of polysaccharides (Gurpilhares, Cinelli, Simas, Pessoa, & Sette, 2019; Song et al., 2018). There is a need for further work to develop a safe, efficient, feasible, and environmentally sound and sustainable method to degrade polysaccharides. Hydrogen peroxide (H2O2, a strong oxidant) could serve as a source of reactive oxygen species (ROS). It is used in the treatment of polysaccharides (such as starch, cellulose and hemicellulose) (Qin, Du, & Xiao, 2002). It is relatively easy to handle, easily available and environmentally friendly, since it decomposes to water and oxygen (Ayanda, Nelana, & Naidoo, 2018). However, treatment with H2O2 does not generally depolymerize high MW polysaccharides, so it is necessary to activate H2O2 to form highly reactive hydroxyl radicals to enhance its depolymerization abilities. Several methods have been developed
⁎ Corresponding author. Present address: School of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou, Guangdong, 510640, China. E-mail address: [email protected] (L. You).
https://doi.org/10.1016/j.carbpol.2019.115647 Received 27 September 2019; Received in revised form 8 November 2019; Accepted 18 November 2019 0144-8617/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Xiaoyong Chen, et al., Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2019.115647
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such as H2O2/ascorbic acid (Mou, Wang, Li, Qi, & Yang, 2018); Fe2+/ ascorbic acid/H2O2 (Zhang et al., 2014); H2O2/ascorbic acid with ultrasound (Shen et al., 2019); SPP (a solution plasma process) with irradiation/H2O2 (Wu et al., 2018); bicarbonate/H2O2 with ultrasound (Hu, Chen, Wu, Zheng, & Ye, 2019); alkaline anthraquinone-2-sulfonate/H2O2 (Hendriks, Kuster, & Marin, 1991); and H2O2/iron sulfophthalocyanine (Klein-Koerkamp et al., 2009). Previous studies indicated that UV/H2O2 mainly relied on the UV (ultraviolet) photolysis of H2O2 to produce hydroxyl radicals, which are strong oxidants that can depolymerize organic compounds, such as benzophenone derivatives (Luo et al., 2019), sulphonamides (Acosta-Rangel, Sánchez-Polo, Polo, Rivera-Utrilla, & Berber-Mendoza, 2018), 3-nitro-1,2,4-trizole-5one (Terracciano et al., 2018) and sulfamethoxazole (Yang et al., 2017). But, there are few studies on the depolymerization abilities of UV/H2O2 with polysaccharides with high MW. Sargassum fusiforme, a brown algae widely distributed in China, Korea, and Japan, has multiple bioactivities, such as anticoagulant, anti-tumor, antioxidant, and regulating immunity (Chen et al., 2012, 2016; Sun et al., 2018). However, due to its dark color, protein content, and high MW, information about processing, structureand bioactivities are limited. Therefore, the depolymerization of polysaccharides with UV/H2O2 at different concentration of H2O2 was studied and compared with UV and H2O2 alone. The effects of UV/H2O2 on physicochemical and structural properties of PSF were also studied along with the in vitro digestion through changes of MW.
Fig. 1. Schematic diagram of UV irradiation processing system.
A second experiment used different PSF concentrations (5.0, 7.5 and 10.0 mg/mL). The optimum condition (UV combined with 150 mmol/L H2O2) was used for the further studies. As controls, the effects of PSF with no treatment, UV treatment, and H2O2 treatment were also studied. 2.3. Determination of MW The MW of PSF before and after UV/H2O2 treatment was measured using high-performance gel permeation chromatography (HPGPC) using a Waters instrument (Model 2414, Waters Co., Milford, MA, USA) equipped with TSK-GEL G-6000 PWXL column (7.8 mm × 300 mm i.d., 13 μm, Tosoh Co, Tokyo, Japan) and TSK-GEL G-3000PWXL column (7.8 mm × 300 mm i.d., 7 μm, Tosoh Co, Tokyo, Japan) connected in series, eluted with 0.02 moL/L KH2PO4 at a flow rate of 0.5 mL/min. The column temperature was kept at 35 ± 0.1 °C. Dextran (National Institute of Metrology, Beijing, China) was used as a MW reference (4.32, 12.6, 126 and 289 kDa). A 25 μL sample was used for each run.
2. Materials and methods 2.1. Preparation of polysaccharides from Sargassum fusiforme (PSF) Sargassum fusiforme were collected from the Dongtou District, Wenzhou, Zhejiang, China. After removing the unwanted debris with tap water, the alga was dried using an air oven at 50 °C for 24 h. The dried alga was ground into powder using an herbal pulverizer (FW177, Tianjin Taisite Instrument Co. Ltd., Tianjin, China) and sieved with a 0.45 mm mesh sieve. This was the coarse ground powder (CGP). Ultrafine powder was obtained by further grinding CGP using an ultrafine pulverizer (XDW6-BI, Jinan Tatsu Micro Machinery Co. Ltd., Jinan, Shandong, China). PSF was extracted using a previous method with some modifications (Ji et al., 2017). Briefly, 100 g ultrafine powder was refluxed three times with 400 mL 95 % ethanol with mild boiling to remove lipids, pigments and low MW compounds. The mixture was centrifuged at 8000 r/min for 15 min at 4 °C (GL21 M, Changsha Xiangzhi Centrifuge Instrument Co. Ltd., Changsha, Hunan, China), and the residue was collected and dried at 50 °C for 12 h. The extraction was carried out at 100 °C for 4 h with a solid to liquid ratio of 1:50 (w/v). The residue was removed using filtration and centrifugation, and the extracted solution was concentrated using evaporation under reduced pressure at 55 °C. The polysaccharide precipitation was carried out using 95 % ethanol to obtain a final ethanol concentration of 80 %. The mixture was kept at 4 °C overnight, and the precipitate was obtained after centrifugation at 8000 r/min for 20 min. The polysaccharide precipitate was washed with 95 % ethanol, and then dried at room temperature. PSF was obtained after the precipitate was freeze-dried (Alpha 1–2 LD plus, Martin Christ Gefriertrocknungsanlage GmbH, Osterode am Harz, Germany).
2.4. Structural characteristics analysis 2.4.1. Determination of polysaccharides, reducing sugar and protein content The total sugar content was determined using the phenol-sulfuric acid method with glucose as a standard (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). The protein content was measured using the Bradford method with bovine serum albumin as a standard (Bradford, 1976). Reducing sugar was measured using the 3, 5-dinitrosalicylic acid method (Lindsay, 1973). 2.4.2. Monosaccharide composition analysis The monosaccharide composition of a sample was determined as previously reported (Ye et al., 2018). Briefly, a 5 mg sample was hydrolyzed using 10 mL 90 % formic acid at 100 °C for 1 h, and then dried using vacuum rotary evaporation at 60 °C. The residue was hydrolyzed again using 10 mL 2 mol/L trifluoroacetic acid at 100 °C for 2 h. The acid was eliminated using repetitive vacuum rotary evaporation with methanol. Then the residue was reduced with hydroxylamine hydrochloride at 90 °C for 30 min after dissolving in 2 mL pyridine and adding 1 mg inositol, and acetylated using 2 mL acetic anhydride at 90 °C for 30 min. The reaction was stopped with 2 mL deionized water. Finally, the acetylated derivatives were extracted twice with 2 mL methylene chloride and the organic phase was collected. Residual pyridine was removed with an equal volume of deionized water, and excess water was removed with anhydrous sodium sulfate. The final productwas filtered through 0.22 μm microporous filtering film and analyzed using Agilent 6890 N gas chromatography (Agilent technologies, Atlanta, GA, USA) with a HP-5 MS capillary column (30 m × 0.32 mm × 0.25 μm, Hewlett Packard Co, Idaho, USA) and a flame ionization detector. A gradient temperature program was used as follows: The injection temperature was 250 °C, the detector temperature was 300 °C, the column
2.2. Degradation of PSF with UV/H2O2 H2O2 was added into 2.5 mg/mL PSF solutions at an initial concentration of 5, 15, 25, 50, 75, 100, and 150 mmol/L. These systems were UV irradiated while the solution was stirred at 150 r/min. The UV irradiation system is shown in Fig. 1. An average irradiance of 6500 mJ/cm2 at room temperature was used. Any remaining H2O2 was decomposed with manganese dioxide, and the manganese dioxide removed using high-speed centrifugation. 2
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determined from 0.1–100 s−1. Samples were individually loaded and allowed to stand for 2 min prior to testing. All measurements were done at 25 ± 0.1 °C.
temperature was programmed from 100 °C for 0.5 min, increasing to 140 °C at 20 °C/min, holding for 5 min, and then increasing to 220 °C at 2 °C/min, finally increasing to 300 °C at 40 °C/min and holding for 3 min. The injection volume was 1 μL. Arabinose, fucose, galactose, glucose, mannose, xylose, and rhamnose were used as the monosaccharide standards (Sigma-Aldrich Chemie Gmbh, Steinhein, Germany). The monosaccharide peaks were identified and quantified by comparison and calibration with the GC profile of the monosaccharide standards.
2.5. Simulated gastrointestinal digestion in vitro 2.5.1. Preparation of gastric and small intestinal solutions Simulated gastric electrolyte solution was prepared according to previous study (Tedeschi, Clement, Rouvet, & Valles-Pamies, 2009). Simulated gastric juice consisted of 37.5 mg of gastric lipase and 35.4 mg of pepsin from porcine gastric mucosa with 150 g of gastric electrolyte solution and 1.5 ml of CH3COONa (1 mol/L, pH 5). The pH of the simulated gastric electrolyte solution and simulated gastric juice were adjusted to pH 3 using 0.1 mol/L HCl. Simulated small intestinal electrolyte solution was prepared according to the same study. Simulated intestinal juice consisted of 20 mL of simulated small intestinal electrolyte solution, 20 g of pancreatin solution (7 % w/w), 40 g bile salt solution (4 % w/w), and 2.6 mg of trypsin. The pH of the simulated intestinal juice was adjusted to 7.5 using 0.1 mol/L NaOH.
2.4.3. Helix-coil transition analysis The conformational structures of the polysaccharides in the solution were determined by characterizing the Congo red-polysaccharide complexes (Qiao et al., 2010). The transition from a triple-helical arrangement to a single-stranded conformation was obtained by measuring the λmax of the Congo red-polysaccharide solutions with NaOH concentrations ranging from 0 to 0.4 mol/L. Polysaccharide aqueous solutions (1 mg/mL) containing 100 μL 80 mmol/L Congo red were mixed with different concentrations of NaOH. The visible absorption spectrum was obtained using a 752 N spectrophotometer (Shanghai Precision and Scientific Instrument Co., Ltd, Shanghai, China) in the range of 400−800 nm at each alkali concentration, with a wavelength interval of 2 nm.
2.5.2. Gastrointestinal digestion in vitro A sample was added to the gastric electrolyte solution and the simulated gastric juice separately to a final concentration of 2 mg/mL. Then the two solutions were combined and digested for 4 h (37 °C, 150 r/min). Digestive juice (5 mL) was collected at 0 and 4 h, inactivated in boiling water for 10 min, and the pH adjusted to 7 using 0.1 mol/L NaOH, and then freeze-dried. Then, the MW change was measured using the HPLC. The rest of the digestive juice was mixed with the simulated intestinal juice at a 10:3 ratio, and its pH adjusted to 7 using 0.1 mol/L NaOH. The mixture was digested for 6 h (37 °C, 150 r/min) and 5 mL digestive juice collected at 0 and 6 h, and inactivated in boiling water for 10 min, and then freeze-dried. The MW changes were measured using HPLC.
2.4.4. I2-KI analysis Sample solution (2 mL) was placed in a test tube, and then 1.2 mL I2KI (containing 0.02 % I2 and 0.2 % KI solution) was added and mixed (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018). The color change of the mixed solution was observed, and their UV–vis spectrum was measured in the range of 300−700 nm, with a wavelength interval of 5 nm. Distilled water was used as the blank. 2.4.5. Fourier transform infrared (FT-IR) spectroscopy The Fourier transform infrared (FT-IR) spectra were obtained using a FT-IR spectrophotometer (Tensor 27, Bruker Co. Ltd., Bergisch Gladbach, Germany). Samples were pressed as KBr pellet and measured in the range of 400–4000 cm−1 (Yu et al., 2019).
2.6. Statistical analysis
2.4.6. Nuclear magnetic resonance (NMR) A sample was dissolved in 99 % deuterium oxide (D2O), then 1H NMR and 13C NMR spectra were obtained with a Bruker AVANCE III HD 600 spectrometer (Bruker Biospin AG, Fallanden, Switzerland, Germany). Data was analyzed using the MestReNova 12 software (MestreLab Research, Santiago de Compostela, Spain). Chemical shifts were given in ppm (Zhang et al., 2016).
Unless otherwise specified, data were expressed as the mean ± SD and were analyzed with Graphpad 7.0 (GraphPad Software, La Jolla, California, USA).
2.4.7. X-ray diffraction (XRD) The crystal structure of powdered samples was done at room temperature using an X-ray diffractometer (SmartLab 3, Rigaku, Tokyo, Japan). The patterns were collected at 2θ diffraction angle from 5° to 90° (Kolsi et al., 2016).
After 2 h with different H2O2 concentration, the MW of the products was measured (Fig. 2). High concentration of H2O2 with UV improved the degradation of PSF in the range of 5−150 mmol/L H2O2 in a concentration dependent manner up to 75 mmol/L and then became relatively steady. However, with UV/150 mmol/L H2O2, samples were also decolorized and deproteinized (Fig. 3B and C). The latter was selected for further study and is identified as LPSF. The degradation of PSF decreased gradually with increasing PSF concentration. When UV or 150 mmol/L H2O2 were used, no obvious changes were observed compared with no treatment, showing the synergistic effect of the combined treatment. Previous studies suggest this was due to the production of many hydroxide free radicals using UV (Acosta-Rangel et al., 2018).
3. Results and discussion 3.1. Effect of UV/H2O2 on MW of PSF
2.4.8. Atomic force microscopy (AFM) A sample was dissolved in deionized water, then diluted to 10−5 mg/mL. After being filtered through a 0.22 μm membrane, 2 μL of the solution was deposited onto a freshly cleaved mica surface and dried at room temperature. AFM was applied using the tapping-mode. Images were acquired by using a Nanodrive Controller (Innova, Veeco, Santa Barbara, CA, USA). Images were obtained using the SPIP software (Version 6.7.8, Image Metrology ApS, Lyngby, Denmark).
3.2. Total sugar, reducing sugar content, protein content and color change of PSF and LPSF
2.4.9. Rheological measurements The rheological properties of samples were done using a HAAKE MARS III rheometer (Thermo Scientific Instruments, Inc., Karlsruhe, Germany) fitted with a serrated plate-plate sensor (60 mm diameter, gap: 1 mm). The apparent viscosity as a function of the shear rate was
The total sugar, reducing sugar and protein contents of PSF and LPSF are summarized in Fig. 3A, B and C. After the UV/150 mmol H2O2 treatment, the total sugar and protein decreased, and reducing sugar 3
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Fig. 2. The MW of PSF with and without UV/H2O2 treatment.
increased. Therefore, the UV/H2O2 treatment had a high recovery rate of total sugar (93.54 %) and clearance rate of protein (76.34 %). The color of LPSF became lighter (Fig. 3D). The change was mainly due to the hydroxide free radical oxidation.
by measuring the wavelength shift of the maximum absorption when Congo red was added to the polysaccharide solutions. In general, if polysaccharides had a triple-helix conformation, the maximum absorption wavelength increased initially and then decreased with increasing NaOH concentration. If there was no trend, those polysaccharides did not have any triple-helix conformation (Qiao et al., 2010). Previous studies have indicated that curdlan was one of the typical polysaccharides with a triple-helix conformation (Chuah, Sarko, Deslandes, & Marchessault, 1983). As shown in Fig. 5A, the maximum absorption wavelength changes of curdlan, PSF and LPSF at various concentrations of NaOH are shown. Results indicated that curdlan and PSF had a triple-helix conformation. However, the maximum absorption wavelength of LPSF decreased gradually with increasing NaOH. Therefore, it had no triple-helical conformation.
3.3. Monosaccharide composition change The monosaccharide composition of PSF and LPSF is shown in Fig. 4A. All monosaccharide found with PSF were also found with LPSF, which suggested that the UV/H2O2 system did not change the type of monosaccharides. However, the molar percentage changed after treatment. Specifically, the molar percentages of fucose (the major sugar present) and xylose decreased, while the molar percent of mannose, glucose and galactose increased. This suggested that fucose and xylose residues were the main active sites for degradation of PSF during the UV/H2O2 treatment.
3.5. I2-KI Previous studies indicated that polysaccharides with fewer branches and shorter side chains could form complexes with I2, and the complexes had an absorption peak at 565 nm. Those with more branches
3.4. Helix-coil transition change The triple-helix conformation of polysaccharides could be evaluated 4
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Fig. 3. The change of total sugar content (A), reducing sugar content (B), protein content (C) and color change (D).
(0.1-10 s−1) than higher (10-100 s−1). In addition, PSF had a higher viscosity (34.99 mPa·s) compared with LPSF (25.32 mPa·s). Previous studies showed that the viscosity was closely related to the MW, so the results (PSF had a higher viscosity) was consistent with the MW results. The shear thinning behavior of PSF and LPSF aqueous solutions was fitted to a power-law model (τ= Kγn, τ is the shear stress; K is the consistency index (Pa·Sn); γ is the shear rate (s−1), and n is the flow behavior index). K and n were determined using the flow curve. Fitting gave an n value for PSF (n = 0.969) and LPSF (0.896) that was < 1, so both were pseudoplastic fluid, with LPSF showing more pseudoplasticity. The K value of PSF (K = 0.012) was greater than the K value of LPSF (K = 0.002), as the viscosity of PSF was higher. Therefore, UV/ H2O2 could reduce the consistency coefficients, and increase the flow characteristic index. The shear thinning behavior could also be observed from the change of stress shown in Fig. 6A (thumbnail). The stress on PSF and LPSF solutions rises with the increase of shear rate, again suggesting shear thinning.
and longer side chains did not. In addition, I2 has been used to detect the presence of starch. If there is no chromogenic reaction, this would suggest no starch in a sample (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018). As shown in Fig. 5B, there was no absorption peak at 565 nm and no chromogenic reaction in solutions of PSF and LPSF. These results suggested no starch and more branches and longer side chains. 3.6. Rheological properties The flow behavior of PSF and LPSF at a concentration of 3 % was measured at 25 °C. Shear thinning occurred due to the orientation of the long polymer molecular chains in the flow field. The randomly positioned molecules became more aligned with the flow direction as the shear rate increased, resulting in fewer interactions between adjacent molecular chains. As shown in Fig. 6A, the apparent viscosities of PSF and LPSF in aqueous solution decreased with increased shear stress, so both were shear thinned pseudoplastic fluids (Wang, Zhao et al., 2019). And the shear thinning was more pronounced at the lower shear rate
Fig. 4. Monosaccharide composition of PSF and LPSF, (A) Red line: standard sample, Gray line: PSF, Green line: PSF with UV/150 mmol H2O2 treatment. (B) Molar percentage of PSF and LPSF (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 5
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Fig. 5. Maximum absorption wavelengths of Congo red with PSF and LPSF mixtures at different NaOH concentrations (A); UV–vis spectrum of PSF and LPSF in the presence of I2-KI (B) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.7. FT-IR spectra
shown), which could be due to its larger MW and complicated components. The 1H and 13C NMR of LPSF are shown in Fig. 7.The 1H NMR spectrum of LPSF (Fig. 7A) showed that multiple peaks were present in the range 4.50–5.50 ppm except the peak at 4.70 ppm, which indicated that LPSF had many kinds of proton signals, and was composed of multiple monosaccharides (Zhang et al., 2016). The result was consistent with the determination of monosaccharide composition. The signals at 3.00–4.00 were heavily overlapped, which may be due to shielding by the hydroxyl groups. Previous studies indicated that the broad signal at 3.50–4.00 ppm was attributed to presence of galactose residues (Raguraman et al., 2019). The signal at 1.33 ppm was attributed to the protons of the CH3 group of the rhamnose residues (Li et al., 2012). The proton signal at 4.56 ppm could be assigned to β-glucuronic acid (Cong et al., 2016). There was no proton signal at 5.40 ppm, indicating that LPSF was composed of glucopyranose (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018), and this was also consistent with the FT-IR analysis. The 13C NMR spectrum (Fig. 7B) showed that multiple peaks were also present in the range 95.00–110.00 ppm, which indicated that LPSF was composed of different monosaccharides (Hu et al., 2016). The signal at 175.46 ppm was assigned to the carboxyl group of glucuronic acid, and the signals at 15.78 and 15.31 ppm were assigned to the methyl groups of rhamnose and fucose. (Hu et al., 2016; Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018) The signals at 70.8–80.8 were assigned to the C-2C-5 of the glycosidic rings (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018). The absence of signals at 83–88 ppm is characteristic of furanosides and is consistent with monosaccharide residues being in the pyranosidic form (Kokoulin et al., 2018).
The FT-IR spectra of PSF and LPSF are shown in Fig. 6B. PSF and LPSF had similar spectral bands except for the peak at 1038 cm−1. Previous studies showed that the peak at 3420−3500 cm−1 corresponds to an OeH stretching vibration, and 1030−1147 cm−1 corresponds to a CeO stretching vibration and OeH bending vibration. These are the main characteristic peaks of polysaccharides (Barker, Bourne, Stacey, & Whiffen, 1954). In addition, peaks at 3433 and 1038 cm−1were observed, and have not been identified. However, the peak intensity of LPSF at 1038 cm−1 was reduced. This was principally due to the effects of hydroxyl radicals. The absorption at 2929 cm−1 corresponds to a eCH3 stretching vibration. The absorptions at 1610 and 1418 cm−1 correspond to a C]O stretching vibration and CeH bending, respectively (Chen et al., 2019). The peak at 1254 cm−1corresponds to S]O stretching vibration, which suggests that PSF and LPSF have sulfate groups (Nakagawa, Asakawa, & Enomoto, 1988). In addition, a weaker peak at 817 cm−1 suggested that the sulfate group was link to the C2 or C3 of PSF and LPSF. The absorption at 889 cm−1corresponded with a β-type glycosidic linkages (Wang, Zhang, Yao, Zhao, & Qi, 2013). The two bands in the region of 1086 and 1038 cm−1 corresponded to the presence of pyranose. Therefore, these results suggest that PSF and LPSF were sulfated polysaccharides, and contained β-type glycosidic linkages with a pyranose ring (Hajji et al., 2019). 3.8. 1H NMR and The 1H and
13
13
C NMR analysis
C NMR spectrum of PSF were abnormal (data not
Fig. 6. Flow behavior (A) and FT-IR spectra (B)of PSF and LPSF. 6
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Fig. 7. 1H (A) and
13
C (B) NMR spectra of LPSF.
3.9. XRD analysis The XRD patterns of PSF and LPSF were observed in the range of 5°90° (Fig. 8). The peak shape trends of PSF and LPSF were different suggesting different structures (Fig. 3D). PSF had a wider and stronger diffraction peak at 22.05° of 2θ, suggesting that PSF had greater crystallinity. LPSF had more diffraction peaks, but their intensities were all weaker. The increase of the diffraction peaks after UV/H2O2 treatment and the degradation of PSF were probably due to the formation of hydrogen bonding of the hydrophilic sites of the fragments. These results were consistent with previous studies (Hu et al., 2019). These results also indicated that UV/H2O2 could lead to a loss of internal PSF crystalline structure. Fig. 8 XRD characterization of PSF and LPSF 3.10. AFM images
Fig. 8. XRD characterization of PSF and LPSF.
AFM is used to observe the surface morphology and roughness of biomacromolecules including polysaccharides (Wang, Cao, Zhang, & Chen, 2019). The AFM images of PSF and LPSF are shown in Fig. 9. There were some apparent differences in the structure of PSF (Fig. 9A)
and LPSF (Fig. 9C) with the structure of LPSF being smaller and more uniform than PSF. The height of protrusions in PSF (Fig. 9B) were higher than that of LPSF (Fig. 9D), indicating that the surface roughness 7
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Fig. 9. AFM planar and 3-dimensional images of PSF (A, B) and LPSF(C, D).
of PSF was greater. These results may be attributed to a decrease in MW as PSF became smaller, and was consistent with UV/H2O2 being an efficient way to degrade PSF.
4. Conclusions Polysaccharides from Sargassum fusiforme were used to study UV/ H2O2 depolymerisation, and the structure and in vitro digestion characteristics. A preparation method for polysaccharides from Sargassum fusiforme with low MW was developed using 150 mmol/L H2O2 and UV irradiation. The advantages of the UV/H2O2 process included the ability to decolor and deproteinate, while degrading polysaccharides. After treatment with UV/H2O2, the structure of the polysaccharides showed changes, such as lower MW, potential destruction of some monosaccharides. Further optimization of UV type and intensity and the reaction system thickness remains to be done. In addition, the results of simulated gastrointestinal digestion showed that polysaccharides with UV/H2O2 treatment were more easily further digested. Therefore, it could be used as a potential method to prepare functional PSF with low MW, but further purification and safety evaluations are necessary.
3.11. Simulated gastrointestinal digestion of PSF and LPSF The simulated gastrointestinal digestion results of PSF and LPSF are shown in Fig.10. The retention time of PSF did not change before and after gastric digestion in either gastric electrolyte solution (SGES) or gastric juice (SGJ) (Fig. 10A③-⑥) Therefore, low acid, pepsin and gastric lipase had no effect on the PSF. However, LPSF could be degraded in the low acid environment, without any contribution from the pepsin and gastric lipase as the two solutions gave similar results (Fig. 10A⑦⑩). The degradation after heat denaturation of the enzymes was also similar (Fig. 10A① and ②). The simulated intestinal digestion showed that the retention time of the gastric digestion products of PSF and LPSF did not change before and after digestion in simulated intestinal juice (SIJ), but other effects were observed. Specifically, when SGJ was added to SIJ before digestion (0 h), an abnormal peak (retention time ∼40 min) was found (Fig. 10B (1)). After digesting for 6 h, another abnormal peak (retention time ∼25 min) was found (Fig. 10B (2)). Similar results were observed with PSF (Fig. 10B (3)-(6)). The LPSF abnormal peak (retention time < 25 min) was observed at 0 h ((Fig. 10B (7) and (9))), but this abnormal peak of PSF was not observed at 0 h (Fig. 10B (3) and (5)). Their role remains unclear. Although PSF could not be digested by gastrointestinal tract in vitro, LPSF could be. However, its products could not be digested by simulated intestinal juice. These different digestive properties may reflect structural differences. The lower MW should also make LPSF easier to use by gut microbes due to the lower MW components.
Declaration of Competing Interest There are no conflicts to declare. Acknowledgements The work was funded by the National Natural Science Foundation of China (31972011), the Guangzhou Science and Technology Program (201907010035), the Guangdong Special Support Program (2015TQ01N670), the Natural Science Foundation of Guangdong Province (2019A1515011670 and 2016A030312001), the 111 project (B17018) and the Fundamental Research Funds for the Central Universities (2019MS101). Thanks to Professor Joe M. Regenstein from Cornell University for its linguistic assistance during the preparation of this manuscript. 8
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Fig. 10. MW change before and after simulated gastrointestinal digestion, (A) Simulated gastric digestion, ① SGJ (0 h), ② SGJ (4 h), ③ SGES + PSF (0 h), ④ SGJ + PSF (0 h), ⑤ SGES + PSF (4 h), ⑥ SGJ + PSF (4 h), ⑦ SGES + LPSF (0 h), ⑧ SGES + LPSF (4 h), ⑨ SGJ + LPSF (0 h), ⑩ SGJ + LPSF (4 h). (B) Simulated intestinal digestion (1) ② + SIJ (0 h), (2) ② + SIJ (6 h), (3) ④ + SIJ (0 h), (4) ④ + SIJ (6 h), (5) ⑥ + SIJ (0 h), (6) ⑥ + SIJ (6 h), (7) ⑧ + SIJ (0 h), (8) ⑧ + SIJ (6 h), (9) ⑩ + SIJ (0 h), (10) ⑩ + SIJ (6 h).
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Degradation of polysaccharides from Sargassum fusiforme using UV/H2O2 and its effects on structural characteristics Xiaoyong Chena, Ruifen Zhangb, Yizhou Lia,c, Xiong Lia,c, Lijun Youa,c,*, Viktoryia Kulikouskayad, Kseniya Hileuskayad a
School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong, 510640, China Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture and Rural Affairs/ Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou, Guangdong, 510610, China c Overseas Expertise Introduction Center for Food Nutrition and Human Health (111 Center), Guangzhou, Guangdong, 510640, China d Institute of Chemistry of New Materials, National Academy of Sciences of Belarus, Minsk, 220141, Belarus b
ARTICLE INFO
ABSTRACT
Keywords: UV H2O2 Polysaccharides Degradation Sargassum fusiforme
The depolymerization effect of UV/H2O2 on the polysaccharides from Sargassum fusiforme (PSF), a brown algae, were studied. The structural changes of PSF before and after UV/H2O2 treatment were analyzed, and molecular weight changes during in vitro digestion were determined. Results indicated that the molecular weight of PSF was reduced from ∼289 to ∼12.6 kDa within 2 h with UV/150 mmol/L H2O2, and the depolymerization effect of UV/H2O2 was significantly higher than that of UV or H2O2 alone. In addition, the UV/H2O2 treatment had a high recovery rate of total sugar (93.54 %) and clearance rate of protein (76.34 %). The monosaccharide composition showed that UV/H2O2 treatment could increase the mole percentage of mannose (37.44 %) and decrease the mole percentage of fucose (14.88 %). The helix-coil transition, X-ray diffraction (XRD) and atomic force microscopy (AFM) imaging showed that the UV/H2O2 treatment depolymerized PSF. Rheological studies indicated that PSF with UV/H2O2 treatment had lower viscosity. In vitro digestion showed that PSF was minimally digested with the in vitro gastrointestinal tract simulation, but PSF with UV/H2O2 treatment could be digested in the low acid environment in the simulated gastric juice, but was minimally digested with the simulated intestinal juice. This studied suggested that the preparation and application of functional PSF with low molecular weight might be beneficial.
1. Introduction The poor degradation of polysaccharides with high molecular weights (MW) is of concern because of poor solubility and bioavailability (Zhang, Wang, Zhao, & Qi, 2014). Various degradation methods have been proposed, including chemical, physical and biological methods. However, chemical methods (such as acid or alkali) have the advantage of simple operations, but may have serious safety and environmental problems (Ren et al., 2017; Xiao, Sun, & Sun, 2001). Physical methods (such as ultrasound, irradiation, microwave, high pressure microfluidization and pulsed electric field) can reduce environmental pollution, but require specialized equipment, and are difficult to do in an industrial environment (Zhou & Ma, 2006). Biological methods (such as enzymatic hydrolysis and microbial fermentation) can be done using mild reaction conditions, good safety, and low cost
features, but screening of enzymes and microbes that can degrade polysaccharides is a difficult problem due to the complicated structures of polysaccharides (Gurpilhares, Cinelli, Simas, Pessoa, & Sette, 2019; Song et al., 2018). There is a need for further work to develop a safe, efficient, feasible, and environmentally sound and sustainable method to degrade polysaccharides. Hydrogen peroxide (H2O2, a strong oxidant) could serve as a source of reactive oxygen species (ROS). It is used in the treatment of polysaccharides (such as starch, cellulose and hemicellulose) (Qin, Du, & Xiao, 2002). It is relatively easy to handle, easily available and environmentally friendly, since it decomposes to water and oxygen (Ayanda, Nelana, & Naidoo, 2018). However, treatment with H2O2 does not generally depolymerize high MW polysaccharides, so it is necessary to activate H2O2 to form highly reactive hydroxyl radicals to enhance its depolymerization abilities. Several methods have been developed
⁎ Corresponding author. Present address: School of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou, Guangdong, 510640, China. E-mail address: [email protected] (L. You).
https://doi.org/10.1016/j.carbpol.2019.115647 Received 27 September 2019; Received in revised form 8 November 2019; Accepted 18 November 2019 0144-8617/ © 2019 Published by Elsevier Ltd.
Please cite this article as: Xiaoyong Chen, et al., Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2019.115647
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such as H2O2/ascorbic acid (Mou, Wang, Li, Qi, & Yang, 2018); Fe2+/ ascorbic acid/H2O2 (Zhang et al., 2014); H2O2/ascorbic acid with ultrasound (Shen et al., 2019); SPP (a solution plasma process) with irradiation/H2O2 (Wu et al., 2018); bicarbonate/H2O2 with ultrasound (Hu, Chen, Wu, Zheng, & Ye, 2019); alkaline anthraquinone-2-sulfonate/H2O2 (Hendriks, Kuster, & Marin, 1991); and H2O2/iron sulfophthalocyanine (Klein-Koerkamp et al., 2009). Previous studies indicated that UV/H2O2 mainly relied on the UV (ultraviolet) photolysis of H2O2 to produce hydroxyl radicals, which are strong oxidants that can depolymerize organic compounds, such as benzophenone derivatives (Luo et al., 2019), sulphonamides (Acosta-Rangel, Sánchez-Polo, Polo, Rivera-Utrilla, & Berber-Mendoza, 2018), 3-nitro-1,2,4-trizole-5one (Terracciano et al., 2018) and sulfamethoxazole (Yang et al., 2017). But, there are few studies on the depolymerization abilities of UV/H2O2 with polysaccharides with high MW. Sargassum fusiforme, a brown algae widely distributed in China, Korea, and Japan, has multiple bioactivities, such as anticoagulant, anti-tumor, antioxidant, and regulating immunity (Chen et al., 2012, 2016; Sun et al., 2018). However, due to its dark color, protein content, and high MW, information about processing, structureand bioactivities are limited. Therefore, the depolymerization of polysaccharides with UV/H2O2 at different concentration of H2O2 was studied and compared with UV and H2O2 alone. The effects of UV/H2O2 on physicochemical and structural properties of PSF were also studied along with the in vitro digestion through changes of MW.
Fig. 1. Schematic diagram of UV irradiation processing system.
A second experiment used different PSF concentrations (5.0, 7.5 and 10.0 mg/mL). The optimum condition (UV combined with 150 mmol/L H2O2) was used for the further studies. As controls, the effects of PSF with no treatment, UV treatment, and H2O2 treatment were also studied. 2.3. Determination of MW The MW of PSF before and after UV/H2O2 treatment was measured using high-performance gel permeation chromatography (HPGPC) using a Waters instrument (Model 2414, Waters Co., Milford, MA, USA) equipped with TSK-GEL G-6000 PWXL column (7.8 mm × 300 mm i.d., 13 μm, Tosoh Co, Tokyo, Japan) and TSK-GEL G-3000PWXL column (7.8 mm × 300 mm i.d., 7 μm, Tosoh Co, Tokyo, Japan) connected in series, eluted with 0.02 moL/L KH2PO4 at a flow rate of 0.5 mL/min. The column temperature was kept at 35 ± 0.1 °C. Dextran (National Institute of Metrology, Beijing, China) was used as a MW reference (4.32, 12.6, 126 and 289 kDa). A 25 μL sample was used for each run.
2. Materials and methods 2.1. Preparation of polysaccharides from Sargassum fusiforme (PSF) Sargassum fusiforme were collected from the Dongtou District, Wenzhou, Zhejiang, China. After removing the unwanted debris with tap water, the alga was dried using an air oven at 50 °C for 24 h. The dried alga was ground into powder using an herbal pulverizer (FW177, Tianjin Taisite Instrument Co. Ltd., Tianjin, China) and sieved with a 0.45 mm mesh sieve. This was the coarse ground powder (CGP). Ultrafine powder was obtained by further grinding CGP using an ultrafine pulverizer (XDW6-BI, Jinan Tatsu Micro Machinery Co. Ltd., Jinan, Shandong, China). PSF was extracted using a previous method with some modifications (Ji et al., 2017). Briefly, 100 g ultrafine powder was refluxed three times with 400 mL 95 % ethanol with mild boiling to remove lipids, pigments and low MW compounds. The mixture was centrifuged at 8000 r/min for 15 min at 4 °C (GL21 M, Changsha Xiangzhi Centrifuge Instrument Co. Ltd., Changsha, Hunan, China), and the residue was collected and dried at 50 °C for 12 h. The extraction was carried out at 100 °C for 4 h with a solid to liquid ratio of 1:50 (w/v). The residue was removed using filtration and centrifugation, and the extracted solution was concentrated using evaporation under reduced pressure at 55 °C. The polysaccharide precipitation was carried out using 95 % ethanol to obtain a final ethanol concentration of 80 %. The mixture was kept at 4 °C overnight, and the precipitate was obtained after centrifugation at 8000 r/min for 20 min. The polysaccharide precipitate was washed with 95 % ethanol, and then dried at room temperature. PSF was obtained after the precipitate was freeze-dried (Alpha 1–2 LD plus, Martin Christ Gefriertrocknungsanlage GmbH, Osterode am Harz, Germany).
2.4. Structural characteristics analysis 2.4.1. Determination of polysaccharides, reducing sugar and protein content The total sugar content was determined using the phenol-sulfuric acid method with glucose as a standard (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). The protein content was measured using the Bradford method with bovine serum albumin as a standard (Bradford, 1976). Reducing sugar was measured using the 3, 5-dinitrosalicylic acid method (Lindsay, 1973). 2.4.2. Monosaccharide composition analysis The monosaccharide composition of a sample was determined as previously reported (Ye et al., 2018). Briefly, a 5 mg sample was hydrolyzed using 10 mL 90 % formic acid at 100 °C for 1 h, and then dried using vacuum rotary evaporation at 60 °C. The residue was hydrolyzed again using 10 mL 2 mol/L trifluoroacetic acid at 100 °C for 2 h. The acid was eliminated using repetitive vacuum rotary evaporation with methanol. Then the residue was reduced with hydroxylamine hydrochloride at 90 °C for 30 min after dissolving in 2 mL pyridine and adding 1 mg inositol, and acetylated using 2 mL acetic anhydride at 90 °C for 30 min. The reaction was stopped with 2 mL deionized water. Finally, the acetylated derivatives were extracted twice with 2 mL methylene chloride and the organic phase was collected. Residual pyridine was removed with an equal volume of deionized water, and excess water was removed with anhydrous sodium sulfate. The final productwas filtered through 0.22 μm microporous filtering film and analyzed using Agilent 6890 N gas chromatography (Agilent technologies, Atlanta, GA, USA) with a HP-5 MS capillary column (30 m × 0.32 mm × 0.25 μm, Hewlett Packard Co, Idaho, USA) and a flame ionization detector. A gradient temperature program was used as follows: The injection temperature was 250 °C, the detector temperature was 300 °C, the column
2.2. Degradation of PSF with UV/H2O2 H2O2 was added into 2.5 mg/mL PSF solutions at an initial concentration of 5, 15, 25, 50, 75, 100, and 150 mmol/L. These systems were UV irradiated while the solution was stirred at 150 r/min. The UV irradiation system is shown in Fig. 1. An average irradiance of 6500 mJ/cm2 at room temperature was used. Any remaining H2O2 was decomposed with manganese dioxide, and the manganese dioxide removed using high-speed centrifugation. 2
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determined from 0.1–100 s−1. Samples were individually loaded and allowed to stand for 2 min prior to testing. All measurements were done at 25 ± 0.1 °C.
temperature was programmed from 100 °C for 0.5 min, increasing to 140 °C at 20 °C/min, holding for 5 min, and then increasing to 220 °C at 2 °C/min, finally increasing to 300 °C at 40 °C/min and holding for 3 min. The injection volume was 1 μL. Arabinose, fucose, galactose, glucose, mannose, xylose, and rhamnose were used as the monosaccharide standards (Sigma-Aldrich Chemie Gmbh, Steinhein, Germany). The monosaccharide peaks were identified and quantified by comparison and calibration with the GC profile of the monosaccharide standards.
2.5. Simulated gastrointestinal digestion in vitro 2.5.1. Preparation of gastric and small intestinal solutions Simulated gastric electrolyte solution was prepared according to previous study (Tedeschi, Clement, Rouvet, & Valles-Pamies, 2009). Simulated gastric juice consisted of 37.5 mg of gastric lipase and 35.4 mg of pepsin from porcine gastric mucosa with 150 g of gastric electrolyte solution and 1.5 ml of CH3COONa (1 mol/L, pH 5). The pH of the simulated gastric electrolyte solution and simulated gastric juice were adjusted to pH 3 using 0.1 mol/L HCl. Simulated small intestinal electrolyte solution was prepared according to the same study. Simulated intestinal juice consisted of 20 mL of simulated small intestinal electrolyte solution, 20 g of pancreatin solution (7 % w/w), 40 g bile salt solution (4 % w/w), and 2.6 mg of trypsin. The pH of the simulated intestinal juice was adjusted to 7.5 using 0.1 mol/L NaOH.
2.4.3. Helix-coil transition analysis The conformational structures of the polysaccharides in the solution were determined by characterizing the Congo red-polysaccharide complexes (Qiao et al., 2010). The transition from a triple-helical arrangement to a single-stranded conformation was obtained by measuring the λmax of the Congo red-polysaccharide solutions with NaOH concentrations ranging from 0 to 0.4 mol/L. Polysaccharide aqueous solutions (1 mg/mL) containing 100 μL 80 mmol/L Congo red were mixed with different concentrations of NaOH. The visible absorption spectrum was obtained using a 752 N spectrophotometer (Shanghai Precision and Scientific Instrument Co., Ltd, Shanghai, China) in the range of 400−800 nm at each alkali concentration, with a wavelength interval of 2 nm.
2.5.2. Gastrointestinal digestion in vitro A sample was added to the gastric electrolyte solution and the simulated gastric juice separately to a final concentration of 2 mg/mL. Then the two solutions were combined and digested for 4 h (37 °C, 150 r/min). Digestive juice (5 mL) was collected at 0 and 4 h, inactivated in boiling water for 10 min, and the pH adjusted to 7 using 0.1 mol/L NaOH, and then freeze-dried. Then, the MW change was measured using the HPLC. The rest of the digestive juice was mixed with the simulated intestinal juice at a 10:3 ratio, and its pH adjusted to 7 using 0.1 mol/L NaOH. The mixture was digested for 6 h (37 °C, 150 r/min) and 5 mL digestive juice collected at 0 and 6 h, and inactivated in boiling water for 10 min, and then freeze-dried. The MW changes were measured using HPLC.
2.4.4. I2-KI analysis Sample solution (2 mL) was placed in a test tube, and then 1.2 mL I2KI (containing 0.02 % I2 and 0.2 % KI solution) was added and mixed (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018). The color change of the mixed solution was observed, and their UV–vis spectrum was measured in the range of 300−700 nm, with a wavelength interval of 5 nm. Distilled water was used as the blank. 2.4.5. Fourier transform infrared (FT-IR) spectroscopy The Fourier transform infrared (FT-IR) spectra were obtained using a FT-IR spectrophotometer (Tensor 27, Bruker Co. Ltd., Bergisch Gladbach, Germany). Samples were pressed as KBr pellet and measured in the range of 400–4000 cm−1 (Yu et al., 2019).
2.6. Statistical analysis
2.4.6. Nuclear magnetic resonance (NMR) A sample was dissolved in 99 % deuterium oxide (D2O), then 1H NMR and 13C NMR spectra were obtained with a Bruker AVANCE III HD 600 spectrometer (Bruker Biospin AG, Fallanden, Switzerland, Germany). Data was analyzed using the MestReNova 12 software (MestreLab Research, Santiago de Compostela, Spain). Chemical shifts were given in ppm (Zhang et al., 2016).
Unless otherwise specified, data were expressed as the mean ± SD and were analyzed with Graphpad 7.0 (GraphPad Software, La Jolla, California, USA).
2.4.7. X-ray diffraction (XRD) The crystal structure of powdered samples was done at room temperature using an X-ray diffractometer (SmartLab 3, Rigaku, Tokyo, Japan). The patterns were collected at 2θ diffraction angle from 5° to 90° (Kolsi et al., 2016).
After 2 h with different H2O2 concentration, the MW of the products was measured (Fig. 2). High concentration of H2O2 with UV improved the degradation of PSF in the range of 5−150 mmol/L H2O2 in a concentration dependent manner up to 75 mmol/L and then became relatively steady. However, with UV/150 mmol/L H2O2, samples were also decolorized and deproteinized (Fig. 3B and C). The latter was selected for further study and is identified as LPSF. The degradation of PSF decreased gradually with increasing PSF concentration. When UV or 150 mmol/L H2O2 were used, no obvious changes were observed compared with no treatment, showing the synergistic effect of the combined treatment. Previous studies suggest this was due to the production of many hydroxide free radicals using UV (Acosta-Rangel et al., 2018).
3. Results and discussion 3.1. Effect of UV/H2O2 on MW of PSF
2.4.8. Atomic force microscopy (AFM) A sample was dissolved in deionized water, then diluted to 10−5 mg/mL. After being filtered through a 0.22 μm membrane, 2 μL of the solution was deposited onto a freshly cleaved mica surface and dried at room temperature. AFM was applied using the tapping-mode. Images were acquired by using a Nanodrive Controller (Innova, Veeco, Santa Barbara, CA, USA). Images were obtained using the SPIP software (Version 6.7.8, Image Metrology ApS, Lyngby, Denmark).
3.2. Total sugar, reducing sugar content, protein content and color change of PSF and LPSF
2.4.9. Rheological measurements The rheological properties of samples were done using a HAAKE MARS III rheometer (Thermo Scientific Instruments, Inc., Karlsruhe, Germany) fitted with a serrated plate-plate sensor (60 mm diameter, gap: 1 mm). The apparent viscosity as a function of the shear rate was
The total sugar, reducing sugar and protein contents of PSF and LPSF are summarized in Fig. 3A, B and C. After the UV/150 mmol H2O2 treatment, the total sugar and protein decreased, and reducing sugar 3
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Fig. 2. The MW of PSF with and without UV/H2O2 treatment.
increased. Therefore, the UV/H2O2 treatment had a high recovery rate of total sugar (93.54 %) and clearance rate of protein (76.34 %). The color of LPSF became lighter (Fig. 3D). The change was mainly due to the hydroxide free radical oxidation.
by measuring the wavelength shift of the maximum absorption when Congo red was added to the polysaccharide solutions. In general, if polysaccharides had a triple-helix conformation, the maximum absorption wavelength increased initially and then decreased with increasing NaOH concentration. If there was no trend, those polysaccharides did not have any triple-helix conformation (Qiao et al., 2010). Previous studies have indicated that curdlan was one of the typical polysaccharides with a triple-helix conformation (Chuah, Sarko, Deslandes, & Marchessault, 1983). As shown in Fig. 5A, the maximum absorption wavelength changes of curdlan, PSF and LPSF at various concentrations of NaOH are shown. Results indicated that curdlan and PSF had a triple-helix conformation. However, the maximum absorption wavelength of LPSF decreased gradually with increasing NaOH. Therefore, it had no triple-helical conformation.
3.3. Monosaccharide composition change The monosaccharide composition of PSF and LPSF is shown in Fig. 4A. All monosaccharide found with PSF were also found with LPSF, which suggested that the UV/H2O2 system did not change the type of monosaccharides. However, the molar percentage changed after treatment. Specifically, the molar percentages of fucose (the major sugar present) and xylose decreased, while the molar percent of mannose, glucose and galactose increased. This suggested that fucose and xylose residues were the main active sites for degradation of PSF during the UV/H2O2 treatment.
3.5. I2-KI Previous studies indicated that polysaccharides with fewer branches and shorter side chains could form complexes with I2, and the complexes had an absorption peak at 565 nm. Those with more branches
3.4. Helix-coil transition change The triple-helix conformation of polysaccharides could be evaluated 4
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Fig. 3. The change of total sugar content (A), reducing sugar content (B), protein content (C) and color change (D).
(0.1-10 s−1) than higher (10-100 s−1). In addition, PSF had a higher viscosity (34.99 mPa·s) compared with LPSF (25.32 mPa·s). Previous studies showed that the viscosity was closely related to the MW, so the results (PSF had a higher viscosity) was consistent with the MW results. The shear thinning behavior of PSF and LPSF aqueous solutions was fitted to a power-law model (τ= Kγn, τ is the shear stress; K is the consistency index (Pa·Sn); γ is the shear rate (s−1), and n is the flow behavior index). K and n were determined using the flow curve. Fitting gave an n value for PSF (n = 0.969) and LPSF (0.896) that was < 1, so both were pseudoplastic fluid, with LPSF showing more pseudoplasticity. The K value of PSF (K = 0.012) was greater than the K value of LPSF (K = 0.002), as the viscosity of PSF was higher. Therefore, UV/ H2O2 could reduce the consistency coefficients, and increase the flow characteristic index. The shear thinning behavior could also be observed from the change of stress shown in Fig. 6A (thumbnail). The stress on PSF and LPSF solutions rises with the increase of shear rate, again suggesting shear thinning.
and longer side chains did not. In addition, I2 has been used to detect the presence of starch. If there is no chromogenic reaction, this would suggest no starch in a sample (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018). As shown in Fig. 5B, there was no absorption peak at 565 nm and no chromogenic reaction in solutions of PSF and LPSF. These results suggested no starch and more branches and longer side chains. 3.6. Rheological properties The flow behavior of PSF and LPSF at a concentration of 3 % was measured at 25 °C. Shear thinning occurred due to the orientation of the long polymer molecular chains in the flow field. The randomly positioned molecules became more aligned with the flow direction as the shear rate increased, resulting in fewer interactions between adjacent molecular chains. As shown in Fig. 6A, the apparent viscosities of PSF and LPSF in aqueous solution decreased with increased shear stress, so both were shear thinned pseudoplastic fluids (Wang, Zhao et al., 2019). And the shear thinning was more pronounced at the lower shear rate
Fig. 4. Monosaccharide composition of PSF and LPSF, (A) Red line: standard sample, Gray line: PSF, Green line: PSF with UV/150 mmol H2O2 treatment. (B) Molar percentage of PSF and LPSF (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 5
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Fig. 5. Maximum absorption wavelengths of Congo red with PSF and LPSF mixtures at different NaOH concentrations (A); UV–vis spectrum of PSF and LPSF in the presence of I2-KI (B) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.7. FT-IR spectra
shown), which could be due to its larger MW and complicated components. The 1H and 13C NMR of LPSF are shown in Fig. 7.The 1H NMR spectrum of LPSF (Fig. 7A) showed that multiple peaks were present in the range 4.50–5.50 ppm except the peak at 4.70 ppm, which indicated that LPSF had many kinds of proton signals, and was composed of multiple monosaccharides (Zhang et al., 2016). The result was consistent with the determination of monosaccharide composition. The signals at 3.00–4.00 were heavily overlapped, which may be due to shielding by the hydroxyl groups. Previous studies indicated that the broad signal at 3.50–4.00 ppm was attributed to presence of galactose residues (Raguraman et al., 2019). The signal at 1.33 ppm was attributed to the protons of the CH3 group of the rhamnose residues (Li et al., 2012). The proton signal at 4.56 ppm could be assigned to β-glucuronic acid (Cong et al., 2016). There was no proton signal at 5.40 ppm, indicating that LPSF was composed of glucopyranose (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018), and this was also consistent with the FT-IR analysis. The 13C NMR spectrum (Fig. 7B) showed that multiple peaks were also present in the range 95.00–110.00 ppm, which indicated that LPSF was composed of different monosaccharides (Hu et al., 2016). The signal at 175.46 ppm was assigned to the carboxyl group of glucuronic acid, and the signals at 15.78 and 15.31 ppm were assigned to the methyl groups of rhamnose and fucose. (Hu et al., 2016; Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018) The signals at 70.8–80.8 were assigned to the C-2C-5 of the glycosidic rings (Liu, Yao et al., 2018, Liu, Ge et al., 2018; Liu, Du et al., 2018). The absence of signals at 83–88 ppm is characteristic of furanosides and is consistent with monosaccharide residues being in the pyranosidic form (Kokoulin et al., 2018).
The FT-IR spectra of PSF and LPSF are shown in Fig. 6B. PSF and LPSF had similar spectral bands except for the peak at 1038 cm−1. Previous studies showed that the peak at 3420−3500 cm−1 corresponds to an OeH stretching vibration, and 1030−1147 cm−1 corresponds to a CeO stretching vibration and OeH bending vibration. These are the main characteristic peaks of polysaccharides (Barker, Bourne, Stacey, & Whiffen, 1954). In addition, peaks at 3433 and 1038 cm−1were observed, and have not been identified. However, the peak intensity of LPSF at 1038 cm−1 was reduced. This was principally due to the effects of hydroxyl radicals. The absorption at 2929 cm−1 corresponds to a eCH3 stretching vibration. The absorptions at 1610 and 1418 cm−1 correspond to a C]O stretching vibration and CeH bending, respectively (Chen et al., 2019). The peak at 1254 cm−1corresponds to S]O stretching vibration, which suggests that PSF and LPSF have sulfate groups (Nakagawa, Asakawa, & Enomoto, 1988). In addition, a weaker peak at 817 cm−1 suggested that the sulfate group was link to the C2 or C3 of PSF and LPSF. The absorption at 889 cm−1corresponded with a β-type glycosidic linkages (Wang, Zhang, Yao, Zhao, & Qi, 2013). The two bands in the region of 1086 and 1038 cm−1 corresponded to the presence of pyranose. Therefore, these results suggest that PSF and LPSF were sulfated polysaccharides, and contained β-type glycosidic linkages with a pyranose ring (Hajji et al., 2019). 3.8. 1H NMR and The 1H and
13
13
C NMR analysis
C NMR spectrum of PSF were abnormal (data not
Fig. 6. Flow behavior (A) and FT-IR spectra (B)of PSF and LPSF. 6
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Fig. 7. 1H (A) and
13
C (B) NMR spectra of LPSF.
3.9. XRD analysis The XRD patterns of PSF and LPSF were observed in the range of 5°90° (Fig. 8). The peak shape trends of PSF and LPSF were different suggesting different structures (Fig. 3D). PSF had a wider and stronger diffraction peak at 22.05° of 2θ, suggesting that PSF had greater crystallinity. LPSF had more diffraction peaks, but their intensities were all weaker. The increase of the diffraction peaks after UV/H2O2 treatment and the degradation of PSF were probably due to the formation of hydrogen bonding of the hydrophilic sites of the fragments. These results were consistent with previous studies (Hu et al., 2019). These results also indicated that UV/H2O2 could lead to a loss of internal PSF crystalline structure. Fig. 8 XRD characterization of PSF and LPSF 3.10. AFM images
Fig. 8. XRD characterization of PSF and LPSF.
AFM is used to observe the surface morphology and roughness of biomacromolecules including polysaccharides (Wang, Cao, Zhang, & Chen, 2019). The AFM images of PSF and LPSF are shown in Fig. 9. There were some apparent differences in the structure of PSF (Fig. 9A)
and LPSF (Fig. 9C) with the structure of LPSF being smaller and more uniform than PSF. The height of protrusions in PSF (Fig. 9B) were higher than that of LPSF (Fig. 9D), indicating that the surface roughness 7
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Fig. 9. AFM planar and 3-dimensional images of PSF (A, B) and LPSF(C, D).
of PSF was greater. These results may be attributed to a decrease in MW as PSF became smaller, and was consistent with UV/H2O2 being an efficient way to degrade PSF.
4. Conclusions Polysaccharides from Sargassum fusiforme were used to study UV/ H2O2 depolymerisation, and the structure and in vitro digestion characteristics. A preparation method for polysaccharides from Sargassum fusiforme with low MW was developed using 150 mmol/L H2O2 and UV irradiation. The advantages of the UV/H2O2 process included the ability to decolor and deproteinate, while degrading polysaccharides. After treatment with UV/H2O2, the structure of the polysaccharides showed changes, such as lower MW, potential destruction of some monosaccharides. Further optimization of UV type and intensity and the reaction system thickness remains to be done. In addition, the results of simulated gastrointestinal digestion showed that polysaccharides with UV/H2O2 treatment were more easily further digested. Therefore, it could be used as a potential method to prepare functional PSF with low MW, but further purification and safety evaluations are necessary.
3.11. Simulated gastrointestinal digestion of PSF and LPSF The simulated gastrointestinal digestion results of PSF and LPSF are shown in Fig.10. The retention time of PSF did not change before and after gastric digestion in either gastric electrolyte solution (SGES) or gastric juice (SGJ) (Fig. 10A③-⑥) Therefore, low acid, pepsin and gastric lipase had no effect on the PSF. However, LPSF could be degraded in the low acid environment, without any contribution from the pepsin and gastric lipase as the two solutions gave similar results (Fig. 10A⑦⑩). The degradation after heat denaturation of the enzymes was also similar (Fig. 10A① and ②). The simulated intestinal digestion showed that the retention time of the gastric digestion products of PSF and LPSF did not change before and after digestion in simulated intestinal juice (SIJ), but other effects were observed. Specifically, when SGJ was added to SIJ before digestion (0 h), an abnormal peak (retention time ∼40 min) was found (Fig. 10B (1)). After digesting for 6 h, another abnormal peak (retention time ∼25 min) was found (Fig. 10B (2)). Similar results were observed with PSF (Fig. 10B (3)-(6)). The LPSF abnormal peak (retention time < 25 min) was observed at 0 h ((Fig. 10B (7) and (9))), but this abnormal peak of PSF was not observed at 0 h (Fig. 10B (3) and (5)). Their role remains unclear. Although PSF could not be digested by gastrointestinal tract in vitro, LPSF could be. However, its products could not be digested by simulated intestinal juice. These different digestive properties may reflect structural differences. The lower MW should also make LPSF easier to use by gut microbes due to the lower MW components.
Declaration of Competing Interest There are no conflicts to declare. Acknowledgements The work was funded by the National Natural Science Foundation of China (31972011), the Guangzhou Science and Technology Program (201907010035), the Guangdong Special Support Program (2015TQ01N670), the Natural Science Foundation of Guangdong Province (2019A1515011670 and 2016A030312001), the 111 project (B17018) and the Fundamental Research Funds for the Central Universities (2019MS101). Thanks to Professor Joe M. Regenstein from Cornell University for its linguistic assistance during the preparation of this manuscript. 8
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Fig. 10. MW change before and after simulated gastrointestinal digestion, (A) Simulated gastric digestion, ① SGJ (0 h), ② SGJ (4 h), ③ SGES + PSF (0 h), ④ SGJ + PSF (0 h), ⑤ SGES + PSF (4 h), ⑥ SGJ + PSF (4 h), ⑦ SGES + LPSF (0 h), ⑧ SGES + LPSF (4 h), ⑨ SGJ + LPSF (0 h), ⑩ SGJ + LPSF (4 h). (B) Simulated intestinal digestion (1) ② + SIJ (0 h), (2) ② + SIJ (6 h), (3) ④ + SIJ (0 h), (4) ④ + SIJ (6 h), (5) ⑥ + SIJ (0 h), (6) ⑥ + SIJ (6 h), (7) ⑧ + SIJ (0 h), (8) ⑧ + SIJ (6 h), (9) ⑩ + SIJ (0 h), (10) ⑩ + SIJ (6 h).
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