Spectrostar nano microplate spectrophotometer

Pure cultures were grown under static conditions at 30°C in 10 mL liquid YEL. After 72 h cells were harvested by centrifugation (14,000 rpm for 3 min), resuspended in sterile water to final OD₆₀₀ = 0.2. For qualitative evaluation of lactose utilization, 24 well microplates containing 990 μL of modified API50CH medium were inoculated with 10 μL of cell suspension prepared as above described. Microplates were incubated under anaerobic conditions (Anaerocult A system, Merck, Darmstadt, Germany) for 7 days at 30°C. The production of acid from lactose was determined based on bromocresol purple color change (de Freitas et al., 2015 (link)). For the evaluation of the kinetics of growth on lactose, cells were inoculated to a final OD₆₅₀ = 0.01 in 200 μL YELactose within 96 well microplates. For the assessment of growth kinetics on lactose plus lactate YELactose was added with increasing concentration of Na lactate (0.0, 1.5, 3.0, and 6.0 g L⁻¹). Growth was measured automatically every hour at OD₆₅₀ using a SPECTROstar nano microplate spectrophotometer (BMG Labtech, Ortenberg, Germany). Carrying capacity (final OD₆₅₀), specific growth rate (μ), lag phase duration (λ), and area under the curve (AUC) were determined by fitting the growth curves with the Bayesian non-parametric model developed by Tonner et al. (2020) (link).

Dose response assays were carried out in 96-well microtiter plates. Aliquots of 135 µL of the cell suspensions containing 10⁴ cells/mL in 20% YPD were mixed in the microtiter plate wells with 15 µL 10× concentrated L-histidine from serial two-fold dilutions. Distilled water was used instead of L-histidine in the control wells. All of the samples were prepared in triplicate. The same test was repeated with the dipeptides histidine–methionine (HM), histidine–valine (HV) and histidine–serine (HS) at ≥95% purity (GenScript, NJ, USA), chosen for being representative of the L-histidine containing dipeptides tested by the PM analysis, and for their different physico-chemical features. The microtiter plates were incubated statically at 30°C for 48 h. Growth was measured automatically every 30 min at OD₆₀₀ using a SPECTROstar nano microplate spectrophotometer (BMG Labtech, Ortenberg, Germany). The average of specific growth rates and the lag time of the curves obtained were analyzed using the DMFit software [26] (link).

Thrombin (Biomed, Lublin, Poland; 0.75 U/ml, in the tris-buffered saline/TBS, pH 7.4) was pre-incubated with alginate nanocomposites for 15 min, at 37 °C. The reaction mixture contained 40 µL of 3 mM Chromogenix S-2238^® substrate (H-D-Phenylalanyl-L-pipecolylLarginine-p-nitroaniline dihydrochloride; Diapharma, Beckett Ridge, OH, USA) and 280 µL of the Thrombin solution, i.e., the control/untreated Thrombin or enzyme pre-incubated with the examined alginate composites. Measurements were executed in 96-well microplates, using a kinetic mode of the BMG Labtech Spectrostar Nano microplate spectrophotometer. Absorbance changes were recorded every 15 s for 10 min, at 415 nm. The hydrolytic activity of Thrombin in all samples was estimated based on the maximal velocity of the reaction (Vmax).