# Characterization and Selection of _Lactobacillus_ Strains with Potential Robotic Applications
## 2 Materials and Methods  
_Lactobacillus strains_  
The strain _Lacticaseibacillus casei_ RC-1 was isolated from homemade cheese from the town of Erzurum, Turkey; the strain _Lactobacillus acidophilus_ RC-2 was isolated from homemade cheese from the town of Edirne, Turkey; and the strain _Lacticaseibacillus rhamn_ RC-10 was isolated from fermented dairy beverage from the town of Etten-Leur, The Netherlands. The strain _Lacticaseibacillus casei_ ATCC 393 was purchased from the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC), Sofia, Bulgaria. The molecular genetic analyses (isolation of total DNA, 165 rDNA PCR amplification, purification of the 165 rDNA and the sequencing of the 165 rRNA gene) of the first three strains (_L. casei_ RC-1, _L. acidophilus_ RC-2 and _L. rhamn_ RC-10) were conducted at the \"Macrogen Europe Laboratory\", The Netherlands. The identification of the strains was performed using the CLC Sequence Viewer software v. 8.0.0. The entire sequence of the 16S rRNA gene was obtained, and the resulting whole sequence was compared with the on-line database sequences via the BLASTn algorithm. Thus, the strains studied were identified to the species level with the corresponding confidence level. The phylogenetic analysis revealed 99% pairwise similarity of the sequence of 16S rDNA of _L. casei_ RC-1 to the partial sequence of 16S rDNA of _L. casei_ strain CAU 6470, 99% similarity of _L. acidophilus_ RC-2 to _L. acidophilus_ strain FSI4, and 99% similarity of _L. rhamn_ RC-10 with _L. rhamn_ strain JCM 1136. All strains were cultured in MRS broth under microaerophilic conditions. **Test microorganisms**  
Twenty microorganisms, including six Gram-positive bacteria (_Bacillus subtilis_ ATCC 6633, _Bacillus cereus_ NCTC 11145, _Staphylococcus aureus_ ATCC 25923, _Listeria monocytogenes_ NBIMCC 8632, _Enterococcus faecalis_ ATCC 19433, _Microococcus luteus_ 2 YC-YT), six Gram-negative bacteria (_Salmonella enteritidis_ ATCC 13076, _Klebsiella pneumoniae_ RC-20, _Escherichia coli_ ATCC 25922, _Proteus vulgaris_ ATCC 6380, _Pseudomonas aeruginosa_ ATCC 9027, _Acineto-bacter baumanii_ RC-22), three yeasts (_Candida albicans_ NBIMCC 74, _Saccharomyces cerevisiae_ ATCC 9763, _Pichia membrananefaciens_) and five fungi (_Aspergillus niger_ ATCC 1015, _Aspergillus ochraceus, Penicillin chrysogenum, Fusarium moniliforme_ ATCC 38932, _Fusarium oxysporum_) from the collection of the Department of Microbiology and Biotechnology at the University of Food Technologies, Plovdiv, Bulgaria, were selected for the antimicrobial activity test. **Culture media**  
_Luria-Bertani agar medium with glucose_  
Luria-Bertani agar medium with glucose (LBG) agar (Condalab SL Madrid, Spain) was used for cultivation of test bacteria. A quantity of 50 g of LBG-solid substance mixture (containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, 10 g glucose and 15 g agar) was dissolved in 1 L of deionized water, pH 7.5 \(\pm\) 0.2. _Malt extract agar_  
Malt extract agar (MEA) (Scharlab SL, Barcelona, Spain) was used for cultivation of test yeasts and fungi. A quantity of 50 g of the MEA-solid substance mixture (containing 30 g malt extract, 5 g mycological peptone and 15 g agar) was dissolved in 1 L of deionized water, pH 5.4 \(\pm\) 0.2. _De Man, Rogosa and Sharpe (MRS) broth and MRS agar_  
These media (Chemsolute, Renningen, Germany) were used for cultivation of lactobacilli. A quantity of 52 g of MRS broth (containing 10 g proteose peptone, 8 g meat extract, yeast 4 g extract, 20 g D(+)-glucose, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 2 g dipotassium phosphate, 1 g polysorbate 80) was dissolved in 1 L of deionized water, pH 6.2 \(\pm\) 0.2. MRS agar was prepared by dissolving 15 g/L of agar (Chemsolute) in MRS broth. The culture media were prepared according to the manufacturer's instructions and autoclaved at 121 \({}^{\circ}\)C for 20 min before use.