Ri 2414

Analysis of lactic acid, sugars and other fermentation products of B. coagulans which may occur such as ethanol and acetic acid was performed using a Waters e2695 HPLC system (Milford USA) equipped with Waters RI2414 and Waters UV/Vis 2489 (measuring at 210 nm) detectors. The column used was a Shodex RS pak KC-811 ion exchange column (length 300 mm, I.D. 8 mm), controlled at 65 °C. As eluent, 3 mM H₂SO₄ in milli-Q water was used. The flow used was 1 mL/min. Samples obtained during fermentation were de-frozen prior to analysis. Two hundred fifty microlitres of this sample was mixed with 250 μL of internal standard, containing 0.25 g/L phthalic acid and 500 μL of milli-Q water. Samples were filtered using 0.2 μm Spartan filters, and supernatants were measured using HPLC.
To determine furan concentrations, UPLC-MS/MS measurements were performed using a Dionex Ultimate 3000 RSLC system, equipped with a Waters Acquity BEH C18 RP column, in combination with a Thermo Scientific^TM LCQ Fleet Ion Trap Mass Spectrometer, as previously described (van der Pol et al. 2015 (link)).

High performance size-exclusion chromatography combined with multi-angle laser light scattering (HPSEC-MALLS) is often used to determine distributions of molecular weight, size and composition independent of column calibration by reference standards [29 (link)].The measurements were conducted on eight-angle laser photometer (Wyatt Technology Co., Santa Barbara, CA, USA) equipped with TSK-GEL G6000 and G4000 PWXL column in 0.5 M NaCl aqueous solution at 38 °C. A differential refractive index detector (RI-2414, Waters, Milford, MI, USA), and ultraviolet detector (UV-2487, Waters, Milford, MI, USA) were simultaneously connected. Meanwhile, an online differential viscometer (ViscoStar™II, Wyatt Technology Co., Goleta, CA, USA) was used to determine the intrinsic viscosity of polysaccharides [30 (link)]. All solutions with a concentration of 5 mg/mL were first centrifuged for 15 min with at 12,000× g, followed by 0.25 μm filter and injected onto the HPLC system. The dn/dc values of samples in aqueous 0.5 M NaCl were determined to be 0.142 mL/g. Huggins and Kraemer plots were used to estimate the intrinsic viscosity [η]. ASTRA 6.1 software (Wyatt Technology, Santa Barbara, CA, USA) was utilized for the data acquisition and analysis [31 (link)].