Uv 1800 spectrophotometer

The reduction of the 1,1′-diphenyl-2-picrylhydrazyl radical (DPPH, Sigma-Aldrich, USA) by Ro was accompanied by photometric measurement in a UV1800 spectrophotometer (Shimadzu, Kyoto, Japan). An amount of 1.5 mL of 40 mM sodium acetate pH 5.5 was mixed into the reaction with 1.0 mL of absolute ethanol containing DPPH to achieve a final concentration of 0.1 mM. After incubation with different Ro concentrations for 5 min at 25 °C, the final absorbance was measured at 517 nm. Quercetin (Q, Sigma-Aldrich, USA) was used as reference. The amount of Fe²⁺ was quantified photometrically at 535 nm using 0.2 mM bathophenanthroline disulfonic acid (Sigma-Aldrich, USA) [50 (link)] in a competitive assay with Ro. At last, the xanthine/xanthine oxidase system was used to generate O₂^•−. The scavenger activity of Ro was estimated by the inhibition of the nitroblue tetrazolium (NBT, Sigma-Aldrich, USA) reduction. After adding 0.08 U/mL xanthine oxidase to a phosphate buffer at a pH of 7.5, which contained 0.05 mM EDTA, 0.2 mM hypoxanthine, and 0.1 mM NBT, the absorbance was measured at 540 nm after 20 min of incubation at 37 °C (Shimadzu UV1800 spectrophotometer, Tokyo, Japan).

An alternative way for measuring the lactate concentration in saliva samples is the colorimetric method of Barker and Summerson, described earlier [21 (link)]. Briefly, 2 mL of glycine-hydrazine buffer (pH 9.5), 0.2 mL of a saliva sample, and 2.2 mL of 0.05 M NAD⁺ were added to a cuvette, and the optical density (E₁) of the solution at 340 nm was recorded with a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) for 5 min at 25 °C. When 0.05 mL of 0.05 M LDH solution was added to the cuvette, the optical density (E₂) of this solution at 340 nm was recorded using the UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) for 5 min at 25 °C. To correct the obtained data, the optical density (E_c) of the solution, with 0.2 mL of saliva sample being changed to 0.2 mL of distilled water, was recorded. The lactate concentration, expressed as μM/g, was calculated according to Equation (4):
where ΔE is the alteration of optical densities of the solution before and after the addition of LDH—(ΔE = (E₂−E₁)−E_c), V is the total volume of the solution (2.45 mL), 6.22 (L/mmol/cm) is the molar extinction coefficient for NADH or NADPH at 340 nm, K is the dilution factor, which is equal to 22.5.

Cell biomass was determined by the optical density at 600 nm (OD_600nm) with a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Glucose was measured by using the SBA-40 biosensor analyzer (Institute of Biology, Shandong Province Academy of Sciences, Shandong, China) equipped with a glucose oxidase membrane. The analytical signal was given by quantifying the production of H₂O₂ generated by glucose oxidation. 5-ALA in the fermentation broth was determined following the method described previously [72 (link)]. Organic acids were analyzed by using HPLC according to the procedure described previously [80 (link)]. The 5-ALA yield was defined as mole of 5-ALA produced/mole of glucose consumed.
The intracellular 5-ALA was measured using the following method. Firstly, the OD_600nm of the culture was detected with a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Secondly, 100 µL 50% glycerol was added into a 1.5 mL centrifuge tube and 200 µL silicone oil was added slowly [81 ]. Then, appropriate amount of fermentation broth was added slowly and then centrifuged at 12,000 rpm for 2 min. Finally, the cells were removed into a clean centrifuge tube and resuspended with adding 20 mM acetic acid. Lysate (200 µL) was taken to determine the intracellular 5-ALA with the method described previously [72 (link)].