Acquity 1 class uplc system

LGG planktonic cultures grown in 50% TSB, TSB supplemented with (1% w/v) D-(+) - glucose, (1% w/v) D-(+)- raffinose or (1% w/v) D- (+)- mannose for 24 h at 37 °C. To get an equal number of bacteria the OD₆₀₀ of the bacteria was compared, and after that washed twice with PBS. For polar metabolite analysis in the polar phase samples, the lyophilized pellets were dissolved using 100 µL DDW-methanol (1:1), centrifuged twice (at maximum speed) to remove possible precipitants, and were injected into LC-MS system. Polar analysis in the polar phase was done as following: Analysis was performed using Waters Acquity I class UPLC System combined with a mass spectrometer (Thermo Exactive Plus Orbitrap) operated in a negative ionization mode. The LC separation was done using the SeQuant Zic-pHilic (150 mm × 2.1 mm) with the SeQuant guard column (20 mm × 2.1 mm) (Merck). The Mobile phase B: acetonitrile and Mobile phase A: 20 mM ammonium carbonate with 0.1% ammonia hydroxide in water: acetonitrile (80:20, v/v). The flow rate was kept at 200 μL min⁻¹ and gradient as follows: 0–2 min 75% of B, 17 min 12.5% of B, 17.1 min 25% of B, 19 min 25% of B, 19.1 min 75% of B, 23 min 75% of B.

Metabolic profiling of the polar phase was performed as described in Zheng et al. (2015) with minor modifications. Briefly, analysis was performed using the Acquity I class UPLC system combined with a mass spectrometer (Thermo Exactive Plus Orbitrap) which was operated in negative ionization mode. The LC separation was performed using SeQuant Zic-pHilic (150 mm×2.1 mm) with the SeQuant guard column (20 mm×2.1 mm) (Merck). Mobile phase A was acetonitrile and mobile phase B was 20 mM ammonium carbonate plus 0.1% ammonia hydroxide in water. The flow rate was kept at 200 μl min⁻¹ and the gradient was: 0–2 min 75% B, 17min 12.5% B, 17.1min 25% B, 19min 25% B, 19.1min 75% B, 19min 75% B.