Ecx400 spectrometer

¹H NMR spectra were recorded using a JEOL ECX400 spectrometer (JEOL, Akishima, Tokyo, Japan). SEM images were obtained using a Hitachi S-4100H electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) at an accelerating voltage of 5 kV. Powder XRD measurements were performed using a PANalytical X’Pert Pro MPD instrument (PANalytical B.V., Almelo, The Netherlands) with Ni-filtered Cu-Kα radiation (λ = 0.15418 nm). Fluorescence spectra were recorded on an FP-6300Q3 spectrometer (JASCO Corporation, Hachioji, Tokyo, Japan).

¹H solution NMR spectra of regenerated SF aqueous solution were observed as a function of Glyc concentration at room temperature by JEOL ECX-400 spectrometer (JEOL Co., Tokyo, Japan).

The ¹H NMR spectra were recorded using a JEOL ECX400 spectrometer (JEOL, Akishima, Tokyo, Japan). The powder X-ray diffraction (XRD) measurements were performed using a PANalytical X’Pert Pro MPD diffractometer (PANalytical B.V., EA Almelo, The Netherlands) with Ni-filtered Cu Kα radiation (λ = 0.15418 nm). The scanning electron microscopic (SEM) images were obtained using a Hitachi S-4100H electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan).

Unless otherwise stated, all operations were performed in a MBraun UNIlab glovebox an argon atmosphere, in a Miwa 1ADB-3KTG glovebox under a nitrogen atmosphere, or by using high-vacuum and standard Schlenk techniques under an argon atmosphere. Benzene (anhydrous) was purchased from Kanto Chemical (Tokyo, Japan) and distilled from benzophenone ketyl prior to use. Other chemicals were purchased from commercial sources and used as received. ¹H-NMR spectra were recorded on a JEOL ECX-500, a JEOL ECX-400, or a JEOL ECS-400 spectrometer (JEOL, Tokyo, Japan), and the chemical shifts of ¹H are referenced to the residual proton signal of CDCl₃ (δ 7.25). ¹³C-NMR spectra were recorded on a JEOL ECX-500 or a JEOL ECX-400 spectrometer (JEOL, Tokyo, Japan), and the chemical shifts of ¹³C are referenced to the signal of CDCl₃ (δ 77.0). All spectra were assigned with the aid of DEPT (distorsionless enhancement by polarization transfer), COSY (correlated spectroscopy), HMQC (heteronuclear multi quantum correlation), and HMBC (heteronuclear multiple bond correlation) NMR experiments. IR spectra were recorded on a JASCO FT/IR-4100 (JASCO, Tokyo, Japan) by utilizing a JASCO ATR Pro550S unit. Melting points were measured with a Yanaco MP-S3 (Yanaco, Tokyo, Japan)and are uncorrected.

The IR spectra were recorded on a PerkinElmer Spectrum Two spectrometer (PerkinElmer Japan Co., Ltd., Kumamoto, Japan). The ¹H NMR spectra were recorded on a JEOL ECX400 spectrometer (JEOL, Akishima, Tokyo, Japan). The viscosity ratees were measured with a Rheosol-G1000 rheometer (UBM, Kyoto, Japan). The DLS measurements were performed on a ELSZ-2000ZS zeta-potential and particle size analyzer (Otsuka Electronics Co., Ltd., Hirakata, Osaka, Japan). The SEM images were obtained using the Hitachi S-4100H electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) by applying a 5 kV accelerating voltage. The TEM images were obtained using the JEOL JEM-3010 electron microscope (JEOL, Akishima, Tokyo, Japan). The TEM samples were prepared by drop-casting aqueous solutions on the carbon-coated copper grid and then staining them with the potassium phosphotungstate solution.

The oligosaccharide S4 was obtained from gum arabic AGP (final concentration, 5.0%) following incubation with the bacterial cell fraction of B. longum subsp. longum JCM7052 grown in larch AGP in 400 mL of 50 mM sodium acetate buffer (pH 5.0) for 53 h. The released oligosaccharides were obtained by ethanol precipitation, and the supernatant was evaporated to dryness. S4 was separated via gel-filtration chromatography on a Bio-Gel P-2 column (φ, 25 by 830 mm; Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with water and high-pressure liquid chromatography using a Cosmosil PBr column (φ, 4.6 by 250 mm; Nacalai Tesque Inc.) and was eluted using 20 mM sodium phosphate (pH 2.5). Finally, the sample was desalted by gel filtration chromatography on a Bio-Gel P-2 column equilibrated with water. The freeze-dried sample was dissolved in deuterium oxide (D₂O). ¹H and ¹³C NMR spectra and two-dimensional spectra (¹H-¹H COSY, HMQC with/without ¹³C-¹H decoupling, HMBC) were measured in D₂O at room temperature on a Jeol ECX 400 spectrometer (400 MHz). For MS, MALDI-TOF mass spectra were recorded on Shimadzu Kompact MALDI Axima-CFR spectrometer with 2,5-dihydroxybenzoic acid as the matrix. ESI-TOF mass spectra were recorded on Jeol AccuTOF JMS-T700LCK with CF₃CO₂Na as the internal standard.

AAm (Nacalai Tesque Inc., Kyoto, Japan), 2-methacryloyloxyethyl phosphorylcholine (MPC) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), N,N’-methylenebis(acrylamide) (bisAA, Tokyo Chemical Industry), and tetraethylene glycol dimethacrylate (TEGMA, Nacalai Tesque) served as monomers and crosslinkers without further purification. The initiator, 2′-azobis [2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044) (Fujifilm Wako Pure Chemical Industries, Osaka, Japan), was utilized without purification. APC was synthesized following a prior report [25 (link)]. Briefly, 2-hydroxyethyl acrylate was reacted with 2-chloro-2-oxo-1,3,2-dioxaphosphorane to produce 2-oxo-1,3,2-dioxaphosphoroyl-oxy-ethyl acrylate, which was then reacted with trimethylamine to yield APC as a white solid. The resulting compound was characterized using ¹H NMR spectroscopy on an ECX-400 spectrometer (JEOL, Ltd., Tokyo, Japan).