Wang et al Anticancer Exopolysaccharide From B breve Iwo1 rude EPS from the culture broth, 1 L of culture samples were was added to 5 mg EPS and was left to stand still. Then 800 centrifuged at 12,000 x g for 15 min at 4@C. After obtaining distilled water was added and hydrolyzed at 100 C for 2.5 h. After the supernatant, TCA was added to a final concentration of cooling, add to 2 mL and neutralize with solid BaCO3. Everything 10% for 12 h at 4oC Precipitated proteins were then removed was centrifuged at 5000 r/min for 10 min to remove impurities, by centrifugation at 4000 x g for 20 min at 4oC. The EPs and remove the supernatant. The supernatant was diluted 20 was precipitated from the supernatant with 3 volumes of times, filtered through a 0. 20 um microporous membrane and cold ethanol followed by an overnight incubation at 4C. subjected to test. After centrifugation at 6000 x g for 30 min at 4oC, the High pH anion exchange chromatography with pulsed pellet containing EPS was resuspended in 2 mL of distilled amperometric detection (HAPEC-PAD) was usedto water and dialyzed(molecular weight cut-off: 6000-8000 Da) determine monosaccharide composition on a DIONEX gainst 1 L of distilled water for 2 days with three water 2500 ion chromatograph system, equipped with on-line changes per 8h. automatic degassing GS50 quaternary gradient pump, Ion exchange chromatography of DEAE Sepharose Fast Flow ED50A electrochemical detector (pulse amperometry (16x 25 mm, GE Healthcare, Amersham, Uppsala, Sweden)was detection), Au working electrode and Ag/AgCl reference used for further EPS purification. Distilled water was applied electrode. Chromatographic conditions were set as follows: to keep the ph of the ion exchange column neutral, eluted sugar column(CarboPac PAl0, 4 x 250 mm) with 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl. The loading volume sugar n column(CarboPac PAl0, 4 x 50 mm) was 1 mL and the flow rate was 1 mL/min. Stepwise collection mobile A. H2O, B 200 mmol/L NaOH, A: B= 91: 9 of 5 mL was applied for each tube. Then, the purified EPs injection volume: 25 uL; flow rate: 1.0 mL/min; column from ion exchange chromatography was further purified by gel temperature: 30C. Chromeleon 6.5 chromatography was filtration chromatography of Sepharose CL-6B(GE Healthcare, used as workstation Amersham, Uppsala, Sweden). Elution was performed with distilled water at a flow rate of 1 mL/min. The final eluted EPs Cell Proliferation and Cytotoxic Analysis was freeze-dried and stored at 4C until analysis A HPLC (Agilent, Santa Clara, CA, United States)equipped examined using CCK-8 assay and DAPI staining. Appropriate ith a NH2 column(Agilent, Santa Clara, CA, United States)was number of cells in 100 HL suspension were plated into 96 used for EPS further purification. The mobile phase consisted well plate and incubated in normoxic or hypoxic environment of 80% ammonium acetate solution and 20% acetonitrile at descripted above. After each time point, 10 uL of the CCK-8 a flow rate of 1.0 mL/min, and the column temperature was solution(Bimake, Houston, TX, United States )was added to each kept on 30 C. The refractive index detector(RID) was used well and incubated for another 4 h at 37 C. The absorbance to detect the eps at 450 nm was determined using a microplate reader. Cell The amount of EPS was determined using the phenol/sulfuric nuclear morphology was stained by DAPI(Zhongshan Golden acid method. Glucose was used as the standard sugar. After Bridge Biotechnology Co., Beijing, China). Each sample had three the color reaction of the glucose standard solution with replicates and each experiment was run in triplicate. phenol/sulfuric acid, its absorbance was measured at 490 nm d the OD490-standard sugar concentration curve was drawn. Western Blot The diluted sample of EPS solution was examined using the After treating cells with EPS, cells were harvested and same color reaction. The 490 nm absorbance value was measured, lysed in RIPA buffer(Applygen Technology, Beijing, China) d the corresponding sugar concentration was found on the containing proteinase inhibitors and phosphatase inhibitors standard curve. The sugar concentration of the sample to be (ROCHe, Basel, Switzerland). BCA kit(Thermo Fisher Scientific, tested was calculated Rockford, IL, United States) was used to measure the protein concentration. Total of 70 ug amounts of protein samples were Scanning Electron Microscopy separated by 10% sodium dodecyl sulfate-polyacrylamide gel Scanning electron microscope was used to examine the electrophoresis(SDS-PAGE) and transferred to polyvinylider morphology of the EPS. Briefly, the freeze-dried EPS powder difluoride(PVDF)membranes by wet blotting. The membranes was mounted on specimens slide, sputter coated with gold and were blocked in 10% non-fat dry milk for I h and then probed examined using SEM(SEM; Hitachi, S-4700, Hitachi Ltd, Tokyo, with antibodies against MCM2 (1: 1,000, ABclonal, Boston, Japan)at accelerating voltages of 10 and 15 kV, respectively MA, United States), Caspases-3(1: 1,000, CST, Boston, MA, United States), PARP(1: 1, 000, CST, Boston, MA, United States), Monomer composition of EPs cleaved-PARP (1: 1, 000, CST, Boston, MA, United States) The monosaccharide standards dried to constant weight were and RPS18(1: 1, 000, ABclonal, Boston, MA, United States) weighed separately, and L-Rha, L-Ara, D-Gal, D-Glc, D-Man, separately at 4C overnight. Followed by incubation with d D-Fru prepared at different concentrations(0.2, 0.5, 1.0, peroxidase-linked secondary antibodies(1: 10,000, CST, Boston, 4.0, and 10.0 ug/mL) were mixed to standard solution. Standard MA, United States) for 1 h at room temperature, the curve for each monosaccharide was drew with monosaccharide enhanced chemiluminescent(ECL) reagent(Thermo Fisher concentration (ug/mL)as the abscissa (x) and peak area Scientific, Rockford, IL, United States)was used to visualize the (nC x min) as the ordinate (y). 200 uL concentrated H2S04 immunoreactive proteins. FrontiersinMicrobiologywww.frontiersin.org May 2019 Volume 10 Article 1044fmicb-10-01044 May 8, 2019 Time: 14:36 # 3 Wang et al. Anticancer Exopolysaccharide From B. breve lw01 crude EPS from the culture broth, 1 L of culture samples were centrifuged at 12,000 × g for 15 min at 4◦C. After obtaining the supernatant, TCA was added to a final concentration of 10% for 12 h at 4◦C. Precipitated proteins were then removed by centrifugation at 4000 × g for 20 min at 4◦C. The EPS was precipitated from the supernatant with 3 volumes of cold ethanol followed by an overnight incubation at 4◦C. After centrifugation at 6000 × g for 30 min at 4◦C, the pellet containing EPS was resuspended in 2 mL of distilled water and dialyzed (molecular weight cut-off: 6000–8000 Da) against 1 L of distilled water for 2 days with three water changes per 8 h. Ion exchange chromatography of DEAE Sepharose Fast Flow (16 × 25 mm, GE Healthcare, Amersham, Uppsala, Sweden) was used for further EPS purification. Distilled water was applied to keep the pH of the ion exchange column neutral, eluted with 0.1, 0.2, 0.3, 0.4, and 0.5 M NaCl. The loading volume was 1 mL and the flow rate was 1 mL/min. Stepwise collection of 5 mL was applied for each tube. Then, the purified EPS from ion exchange chromatography was further purified by gel filtration chromatography of Sepharose CL-6B (GE Healthcare, Amersham, Uppsala, Sweden). Elution was performed with distilled water at a flow rate of 1 mL/min. The final eluted EPS was freeze-dried and stored at 4◦C until analysis. A HPLC (Agilent, Santa Clara, CA, United States) equipped with a NH2 column (Agilent, Santa Clara, CA, United States) was used for EPS further purification. The mobile phase consisted of 80% ammonium acetate solution and 20% acetonitrile at a flow rate of 1.0 mL/min, and the column temperature was kept on 30◦C. The refractive index detector (RID) was used to detect the EPS. The amount of EPS was determined using the phenol/sulfuric acid method. Glucose was used as the standard sugar. After the color reaction of the glucose standard solution with phenol/sulfuric acid, its absorbance was measured at 490 nm, and the OD490-standard sugar concentration curve was drawn. The diluted sample of EPS solution was examined using the same color reaction. The 490 nm absorbance value was measured, and the corresponding sugar concentration was found on the standard curve. The sugar concentration of the sample to be tested was calculated. Scanning Electron Microscopy Scanning electron microscope was used to examine the morphology of the EPS. Briefly, the freeze-dried EPS powder was mounted on specimens slide, sputter coated with gold and examined using SEM (SEM; Hitachi, S-4700, Hitachi Ltd., Tokyo, Japan) at accelerating voltages of 10 and 15 kV, respectively. Monomer Composition of EPS The monosaccharide standards dried to constant weight were weighed separately, and L-Rha, L-Ara, D-Gal, D-Glc, D-Man, and D-Fru prepared at different concentrations (0.2, 0.5, 1.0, 4.0, and 10.0 µg/mL) were mixed to standard solution. Standard curve for each monosaccharide was drew with monosaccharide concentration (µg/mL) as the abscissa (x) and peak area (nC × min) as the ordinate (y). 200 µL concentrated H2SO4 was added to 5 mg EPS and was left to stand still. Then 800 distilled water was added and hydrolyzed at 100◦C for 2.5 h. After cooling, add to 2 mL and neutralize with solid BaCO3. Everything was centrifuged at 5000 r/min for 10 min to remove impurities, and remove the supernatant. The supernatant was diluted 20 times, filtered through a 0.20 µm microporous membrane and subjected to test. High pH anion exchange chromatography with pulsed amperometric detection (HAPEC-PAD) was used to determine monosaccharide composition on a DIONEX- 2500 ion chromatograph system, equipped with on-line automatic degassing GS50 quaternary gradient pump, ED50A electrochemical detector (pulse amperometry detection), Au working electrode and Ag/AgCl reference electrode. Chromatographic conditions were set as follows: sugar analysis column (CarboPacTM PA10, 4 × 250 mm); sugar protection column (CarboPac PA10, 4 × 50 mm); mobile phase: A. H2O, B. 200 mmol/L NaOH, A:B = 91:9; injection volume: 25 µL; flow rate: 1.0 mL/min; column temperature: 30◦C. Chromeleon 6.5 chromatography was used as workstation. Cell Proliferation and Cytotoxic Analysis The EPS effect on cell proliferation and cytotoxicity were examined using CCK-8 assay and DAPI staining. Appropriate number of cells in 100 µL suspension were plated into 96- well plate and incubated in normoxic or hypoxic environment descripted above. After each time point, 10 µL of the CCK-8 solution (Bimake, Houston, TX, United States) was added to each well and incubated for another 4 h at 37◦C. The absorbance at 450 nm was determined using a microplate reader. Cell nuclear morphology was stained by DAPI (Zhongshan Golden Bridge Biotechnology Co., Beijing, China). Each sample had three replicates and each experiment was run in triplicate. Western Blot After treating cells with EPS, cells were harvested and lysed in RIPA buffer (Applygen Technology, Beijing, China) containing proteinase inhibitors and phosphatase inhibitors (ROCHE, Basel, Switzerland). BCA kit (Thermo Fisher Scientific, Rockford, IL, United States) was used to measure the protein concentration. Total of 70 µg amounts of protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes by wet blotting. The membranes were blocked in 10% non-fat dry milk for 1 h and then probed with antibodies against MCM2 (1:1,000, ABclonal, Boston, MA, United States), Caspases-3 (1:1,000, CST, Boston, MA, United States), PARP (1:1,000, CST, Boston, MA, United States), cleaved-PARP (1:1,000, CST, Boston, MA, United States) and RPS18 (1:1,000, ABclonal, Boston, MA, United States) separately at 4◦C overnight. Followed by incubation with peroxidase-linked secondary antibodies (1:10,000, CST, Boston, MA, United States) for 1 h at room temperature, the enhanced chemiluminescent (ECL) reagent (Thermo Fisher Scientific, Rockford, IL, United States) was used to visualize the immunoreactive proteins. Frontiers in Microbiology | www.frontiersin.org 3 May 2019 | Volume 10 | Article 1044