2 nitrophenyl β d galactopyranoside onpg

The β-galactosidase assays were modified from a previously reported study [65 (link)]. Overnight bacterial cultures were diluted 1:500 in TSB. If needed, appropriate amounts of oxidants, iron chelators, or FeCl₃ were included in the medium. The β-galactosidase activity was monitored by collecting samples at different time points. A total of 100 µL of bacterial culture was added to 900 µL of Z Buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7.0, 0.2% β-mercaptoethanol). A total of 1 µL of 0.1% sodium dodecylsulfate (SDS) and 50 µL of chloroform were added to the suspension, which was mixed vigorously for 20 s. The suspension was then incubated for 5 min at 30 °C. A total of 200 µL of 4 mg/mL 2-nitrophenyl β-d-galactopyranoside (ONPG, Sigma) was added to the cells. The reaction was stopped by adding 500 µL of 1 M Na₂CO₃. The suspension was centrifuged at 14,000× g for 3 min, and the optical densities of the supernatant were read at 420 and 550 nm. The β-galactosidase activity was then calculated in Miller Units (MUs) according to the following equation: $$\mathrm{MU}=\frac{1000\times \left({\mathrm{OD}}_{420}-1.75\times {\mathrm{OD}}_{550}\right)}{\mathrm{Time}\left(\min \right)\times \mathrm{Volume}\left(\mathrm{mL}\right)\times {\mathrm{OD}}_{600}}$$

2-aminoanthracene (2AA) and chloropromazine (CPZ) were purchased from Sigma-Aldrich (Poznań, Poland). 2AA were dissolved in DMSO. CPZ was dissolved in deionized water. D-glucose 6-phosphate disodium salt hydrate (G-6-P) (CAS no. 3671-99-6), disodium salt hydrate (CAS no. 3671-99-6), and 2-nitrophenyl β-d-galactopyranoside (ONPG) (CAS no. 369-07-3), which was the β-galactosidase enzyme, were purchased from Sigma Aldrich. Nicotinamide adenine dinucleotide phosphate (NADP) (CAS no. 24292-60-2) was purchased from MP Biomedicals (Irvine, CA, USA). DMSO (CAS no. 67-68-5) was purchased from Avantor Performance Materials (Gliwice, Poland). Methanol (CAS no. 67-56-1) was purchased from Merck (Warsaw, Poland).

V. parahaemolyticus strains containing the empty pBAD vector, pBAD-lacZ-vtrC or pBAD-vtrC-lacZ were grown overnight in MLB at 30°C. Overnight cultures were diluted to OD_600 nm = 0.6 in MLB supplemented with 0.1% arabinose and induced for 5 hr at 30°C. Bacterial cells were permeablized by mixing 20 μl of bacterial culture with 80 μl permeabilization solution (100 mM Na₂HPO₄, 20 mM KCl, 2 mM MgSO₄, 0.8 mg/ml hexadecyltrimethylammonium bromide, 0.4 mg/ml sodium deoxycholate, 5.4 μl/ml β-mercaptoethanol). 600 μl substrate solution (60 mM Na₂HPO₄, 40 mM NaH2PO₄, 1 mg/ml 2-Nitrophenyl β-D-galactopyranoside (ONPG, Sigma-Aldrich N1127), 2.7 μl/ml β-mercaptoethanol) was added to the samples to start the reaction and the time was recorded as T1 (min). Samples were incubated at 30°C until a pale yellow color develops. The reaction was stopped by adding 100 μl 1 M Na₂CO₃ and the time was recorded as T2 (min). The supernatant was collected after centrifugation at 20000 x g for 5 min. Measure the OD_420nm of the supernatant. β-galactosidase activity was calculated as below:
β-galactosidase activity (Miller unit) = 1000* OD_420 nm/0.02* OD_600 nm *(T2-T1)

Different concentrations of baicalein or PB (16 μg/mL) were coincubated with bacterial suspension (OD₆₀₀ = 0.5) for 30 min (37°C). Then, 190 μL of supernatants (12,000 × g, 15 min, 4°C) was incubated with 2-nitrophenyl-β-d-galactopyranoside (ONPG; 3 mmol/L; Sigma) for 30 min, and OD₄₂₀ was detected (53 (link)).