# Evaluation of Non-Encapsulated and Microencapsulated Lactic Acid Bacteria
## 2 Materials and Methods
### Molecular Identification of Lactic Acid Bacteria  
Total genomic DNA of _Lactobacillus_ strains was extracted using a Wizard Genomic DNA Purification Kit (Promega, Corporation, WI, USA) according to the manufacturer's instructions. The quality and quantity were checked using a NanoDrop 8000 spectrophotometer (ThermoFisherScientific, Wilmington, DE, USA). Preliminary characterization of isolated strains was performed based on the phenotype traits. Molecular identification of lactic acid bacteria was done by sequence analysis of a 16S rRNA gene (16S rRNA). Two pairs of primers were used to amplify and sequence different regions of the gene. The forward and reverse primers Lac1 F (5\({}^{\prime}\)-AGCATGGGAATCTTCCA-3\({}^{\prime}\)) and Lac1 R (5\({}^{\prime}\)-ATTYCACCGTACACATG-3\({}^{\prime}\)) were designed accordingly to [PERSON] et al. (2015) with some minor modifications to amplify a 345 bp fragment from variable regions V2-V4 of 16S rRNA gene. A second pair of primers, 27F (5\({}^{\prime}\)-AGAGTTTGATCCGGCTCAG-3\({}^{\prime}\)) and 1492R (5\({}^{\prime}\)-GGTTACCTTTGTTACGACT-3\({}^{\prime}\)) were used to amplify and sequence the complete region of 16S rRNA gene when the sequencing of 345 bp fragment amplified with the primers Lac1F/R showed to be inconclusive. In the both cases, the PCR was performed with an AmpliTaq polymerase kit (Applied Biosystems). Each PCR mixture (25 \(\mu\)L) contained 2.5 \(\mu\)L PCR buffer 10X, 1.5 \(\mu\)L 25 mM MgCl\({}_{2}\) \(\mu\)L dNTPs mix (containing each dNTP at a concentration of 2 mM), 0.5 \(\mu\)L of each primer (with concentration 20 pmols/\(\mu\)L); 0.1 \(\mu\)L of AmpliTaq polymerase (5U/\(\mu\)L) and the corresponding volumes of sterile Milli-Q water and DNA diluted solution.  
Amplification was performed in a Veriti(tm) 96-Well Thermal Cycler (ThermoFisher Scientific) programmed for 10 min at 95 \({}^{\circ}\)C (initial denaturation) and 30 cycles of 30 s at 95 \({}^{\circ}\)C (denaturation), 30 s at 60 \({}^{\circ}\)C (annealing), 1 min at 72 \({}^{\circ}\)C (extension) and 10 min at 72 \({}^{\circ}\)C (final extension). Reaction products were resolved by electrophoresis in 2% (\(w/v\)) agarose gels and visualized by ethidium bromide staining. The sequencing of the PCR products was done by using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Wilmington, DE, USA). The automated sequencing of both strands of the PCR product was done on an ABI Prism 3130 automated gene sequencer (Applied Biosystems). The Basic Alignment Search Tool (BLAST) tool ([[http://blast.ncbi.nlm.nih.gov/Blast.cgi](http://blast.ncbi.nlm.nih.gov/Blast.cgi)]([http://blast.ncbi.nlm.nih.gov/Blast.cgi](http://blast.ncbi.nlm.nih.gov/Blast.cgi)), accessed on 11 October 2021) was used to assess the degree of similarity of our sequences with similar 16S rRNA sequences from the GenBank database. BLAST is an online program belonging to NCBI (National Center for Biotechnology Information) to establish the hits of subjects sequences deposited in the international nucleotide databases (e.g., GenBank, EMBL etc.) giving the best matching with the query sequence ([PERSON] et al., 2013).