Maxq 7000

Escherichia coli K12 strain MG1655 was used in this study. Seed cultures were grown in LB medium (Bio Basic) and used to inoculate pre-cultures in appropriate growth media without antibiotics. After overnight growth, pre-cultures were diluted (500 − 1,000×) to fresh media and allowed to resume exponential growth for at least three generations before being diluted into media containing antibiotics. Cells were adapted to exponential growth in antibiotics and grown in adapted growth for four generations before growth rate measurements were taken. Cells were grown in 3 ml of culture media at 37°C in 20-mm test tubes, shaken in a water bath (MaxQ 7000, Thermo-Fisher) at 250 rpm. Growth rate was monitored by measuring OD₆₀₀ on a Biomate 3S spectrophotometer (Thermo-Fisher) over time, with cell viability corroborated by plating. The translational mutant strain appearing in Fig6 is derived from a mutant exhibiting pseudo-dependence on streptomycin and a corresponding decreased translation rate in the absence of streptomycin (Ruusala et al, 1984 (link); Scott et al, 2010 (link)). The mutation (in rpsL) was moved from strain GQ9 (Scott et al, 2010 (link)) (also known as CH349 or UK317 (Ruusala et al, 1984 (link))) to our background strain (MG1655) via P1 transduction and selection on streptomycin.

The in vitro digestion of LHEPS and LPEPS was performed by our previous methodologies with minor modifications [27 (link)]. The procedure was initiated with a simulated human salivary phase, followed by a gastric phase and an intestinal phase. A pH-meter model 744 (Metrohm AG, Herisau, Switzerland) was applied for monitoring the pH of the digestion solution. For the buccal phase, 10 mg of LHEPS or LPEPS was added to the 4 mL mixture of salivary α-amylase and amyloglucosidase, and 10 mL buffer solution. The digestion solution was carried out in a shaking water bath (MaxQ™ 7000, Thermo Fisher, Waltham, MA, USA) for 10 min (37 °C, 55 rpm). For the gastric phase, the pH of digestion solution was adjusted to 2.0 using 1 M HCl, then pepsin was added to a final concentration of 3.0 mg/mL and incubated at 37 °C for 2 h in darkness while stirring (55 rpm). For the intestinal phase, 4 mL mixture of pancreatin, trypsin, pancreatic α-amylase, and bile salt solution was added and 1 M NaOH was applied to regulate the pH of the digestion solution. The digestion phase was continued at 37 °C, 150 rpm for 8 h. Finally, the mediums were inactivated immediately at 100 °C for 5 min and filtered before analysis. Each in vitro simulated saliva and GSI digestion was conducted three times or more to ensure repeatability.