# Characterization and Selection of _Lactobacillus_ Strains with Potential Robotic Applications
## 2 Materials and Methods
**The effect of temperature on the growth of _Lactobacillus_ strains**  
_Determination of viable cell counts_  
In order to determine the growth characteristics, fresh, 24 h cultures of the _Lactobacillus_ strains (2%) were inoculated in MRS broth. The inoculated medium was transferred in equal portions into sterile tubes, which were incubated at 30 \({}^{\circ}\)C and 37 \({}^{\circ}\)C. Aliquots for determination of the number of viable cells (by spread plating onto MRS agar) and titratable acidity at 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 36 h and 48 h were taken. The number of viable cells was expressed as log CFU/mL. _Determination of titratable acidity_  
The titratable acidity was assessed by titration of 2 mL of sample (diluted with 10 mL of distilled water) with 0.1 N NaOH using phenolphthalein as an indicator until the appearance of a pale pink color persisting over 1 min. The results from three consecutive experiments were expressed as % lactic acid [16]. **Resistance to bile salts**  
The resistance to bile salts was implemented according to [PERSON] et al. [17] with some modifications. Fresh, 24 h culture of the _Lactobacillus_ strain (2%) was inoculated in MRS broth supplemented with 0.25, 0.5, 0.75 and 1% (\(w/v\)) of bile salts (Oxoid, Basingstoke, UK). Samples without addition of bile salts (0%) were used as controls. The samples were transferred in quantity of 200 \(\mu\)L per well in a sterile 96-well microplate with a lid (Sartorius, Helsinki, Finland) and incubated at 37 \({}^{\circ}\)C in an orbital incubator Stuart SI500 (Keison, Chelmsford, UK) with slow shaking (60 rpm). The optical density (OD) was measured at 600 nm at 0 h, 2 h, 4 h, 6 h, 8 h and 24 h of incubation using a microplate spectrophotometer system SpectroStar Nano (BMG Labtech, Ortenberg, Germany). Survival rate (S) was calculated using the following equation:  
\[\%\ S=\frac{ODstrain+bile\ salts}{ODcontrol}\times 100 \tag{1}\]  
**Resistance to simulated upper gastrointestinal tract conditions**  
The resistance of the strains to the gastrointestinal tract conditions was performed by combining the methods of [PERSON] et al. [17] and [PERSON] et al. [3]. Fresh, 24 h cultures of the _Lactobacillus_ strains were pelleted by centrifugation at 5000 rpm for 10 min, triple-washed with phosphate buffer saline (PBS) (pH 7.4) and resuspended in 3 mL of PBS. These suspensions were inoculated at a 1/10 ratio (\(v/v\)) in artificial gastric juice prepared by dissolving pepsin (Fisher Scientific, Geel, Belgium) at concentration of 2 g/L in 0.5% sterile saline previously adjusted to pH 2.5 and artificial small intestinal juice prepared by dissolving pancreatin (Niksen Ltd., Gabrovo, Bulgaria) at concentration of 0.25 g/L in PBS with pH 7.4. The treated suspensions were incubated at 37 \({}^{\circ}\)C for 3 h and then centrifuged, triple-washed with PBS and resuspended in MRS broth. Next, aliquots of 200 \(\mu\)L per well were transferred in a sterile 96-well microplate with a lid (Sartorius). Recovery of the cells was assessed by OD at 600 nm at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 24 h of incubation at 37 \({}^{\circ}\)C with slow shaking (60 rpm) using a microplate spectrophotometer system SpectroStar Nano (BMG Labtech). **Antimicrobial activity assay**  
Antimicrobial activity was determined using the agar well diffusion method as described in our previous research [18]. The antimicrobial activity of the four _Lactobacillus_ strains components--whole _Lactobacillus_ culture (LC); cell biomass (CB) centrifuged at 4500 rpm for 10 min, triple-washed with PBS and resuspended in PBS; cell-free supernatant (CFS) obtained after the centrifugation and filtered through a sterile syringe filter (poresize = 0.45 \(\upmu\)m, Aijinen, China); and neutralized cell-free supernatant (NCFS) to pH = 7.0 with sterile 10% NaOH--was assessed by measuring the diameter of the inhibition zones (IZs) around the wells on the 24 th and 48 th hour of incubation.