Molecular identification, bioinformatics analysis and phylogenetic relationships of Lactobacillus plantarum in traditional and industrial sourdough

Bread is considered as one of the most important commodity which has provided human nutritional needs for several centuries. In recent decades, bread consumption has witnessed tremendous increase due to rising of other foods expenses. Cereal and bread industries has been always concerned about bread production in an industrial scale along with suitable shelf life, acceptable organoleptic properties and free from any chemical additives and preservatives. Among different methods such as addition of synthetic enzymes, gums, emulsifiers and improvers, sour dough has gained special attention besides shelf life enhancement and improvement of sensorial and nutritional features. The main role of sour dough has been prominent for fermentation and gas production in dough as a result of inclusion of Lactic Acid Bacteria (LAB) as a technology. Fluctuation of process parameters including temperature, dough yield and starter culture composition determine the sour dough quality. The main reason of sour dough application in wheat bread is flavor enhancement. Culture-based microbiological examinations have accompanied with some difficulties in accurate and precise identification of Lactobacilli. So, in recent years, in order to identify precisely, different molecular and PCR assays and gene sequencing have been applied. Thus, in present study, fragment of 253bp from 16 S rRNA gene in Lactobacillus plantarum was amplified with the help of PCR and sequenced in the next step. Also, comparison of nucleotide sequence of this region of traditional Lb. plantarum with nucleotide sequence of industrial starter (Germany) and sequences deposited in Gene Bank was evaluated.
Material and Sampling: According to sensory properties, sour dough samples were collected from Torbat e jam city and industrial starter sample (sour dough) was prepared from Buker Company, Germany.
Isolation and bio chemical analysis: Ten- fold serial dilutions of samples were prepared and cultured on MRS agar. Gram positive, catalase negative isolates were considered as presumptive LAB.
Molecular Identification: DNA of isolates were extracted according to the procedure of DNA extraction kit (Thermo, K721, USA). In order to amplify the 253 bp fragment of 16S rRNA in Lb. plantarum, specific primers were designed using Primer Premier 5. PCR was performed in Biometra Thermocycler (T- personal, Germany) in 36 cycles. In the following step, PCR products were sent to Macrogen Company for sequencing.
Bioinformatics analysis: Analysis of sequencing results was conducted with the aid of bioinformatics soft wares including CLC Main work bench, Chromas Lite 2.01, MEGA 5 and BioEdit. Phylogenetic tree was designed among different isolates using Neighbor-Joining method (bootstrap=1000) in MEGA 5 software. For determination of genetic distance, Create Pairwise comparison method was used in CLC Main workbench.
Quality and quantity of extracted DNA samples were confirmed by Gel electrophoresis. PCR and electrophoresis of PCR products confirmed and proved the accuracy and validity of amplified 253 bp fragment with intended primers. Comparison of Torbat and Germany nucleotide sequences demonstrated that two replacement mutations have been occurred including (A/C) and (T/C) in positions 182 and 205, respectively. These changes resulted in replacement of Histidine by Proline, and Glutamine by stop codon. Nucleotide content of 16S rRNA gene sequence in Lb. plantarum isolated from Torbat e Jam and Germany sour dough samples displayed both sequences contained 49.4% (G) and 50.6% (A). Phylogenetic results demonstrated that Lb. plantarum isolated from Torbat e Jam and Germany samples were classified in the same sub group. This implies the close phylogenetic relationship between these two lactobacilli. In order to identify Lb. plantarum, 16S rRNA gene was sequenced in sour dough samples of Germany and Belgium and phylogenetic tree showed that Lb. plantarum had the lowest genetic distance with Lb. pontis and highest one with Lb. sanfranciscensis and Lb. salivarius