MOLECULAR CHARACTERIZATION OF ISOLATED LACTIC ACID BACTERIA FROM DIFFERENT TRADITIONAL DAIRY PRODUCTS OF TRIBES IN THE FARS PROVINCE, IRAN

Various traditional dairy products could be considered as an abundant source for Isolation/collection of new lactic acid bacteria (LAB) with unique characteristics. The present researchaimed to investigate the morphological, biochemical and genotypic characterization of dominant lactic acid bacteria that were isolated from traditional dairy products in Iranian tribes . A total of 75 samples of traditional yogurt, doogh from dairy units in Fars province were randomly collected. Isolation of lactic acid bacteria, biochemical and genotypic identification were conducted. Totally, 157 LAB isolates were selected. Cocci and rod shape LAB were 53.50% and 46.49%, respectively. Biochemical tests showed the occurrence of 40.6% of the strains were Streptococcus, 2.25% of the strains were enterococci and 20.3% of the strains were lactococci. Most isolated lactobacilli were related to Lactobacillus plantarum and Lactobacillus bulgaricus. Genetically the presence of the following species was verified: Lactococcus lactis subsp. cremoris, Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus fermentum, Lactococcus lactis subsp. lactis, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus casei. The current study showed that thestrains which were isolated from traditional dairy products were not only appropriatefor use as starter adjuncts or cultures, but also they may provide a valuable gene pool for research and production of commercial starters with specific traits.

morphology, and oxidase activity. Catalase negative and Gram-positive, nonspore forming, oxidase negative, cocci or rods isolates were selected as presumptive LAB and stored in growth (MRS or GM17 or Tryptic soy broth (TSB)) medium containing 15% (vv -1 ) glycerol as stocks frozen at -80°C.

Biochemical identification
Biochemical identification of the isolated/selected bacteria was carried out following Bergey's manual of determinative bacteriology. (Breed, Murray, & Smith, 1957) For biochemical identification, every single isolate was activated in 5 ml GM17 or MRS or TSB broth medium for 24 h at 37°C prior to use. The tests used for preliminary characterization of isolates included Gram staining, catalase test (H2O2, 3%), oxidase test and endospore formation(spore staining). Isolates with Gram-positive, catalase, and oxidase-negative, non spore forming characteristics were selected for further identification (Nikita & Hemangi, 2012).

Identification of cocci
Inoculated bacterial isolates in GM17 broth medium were incubated at 10°C, 40°C and 45°C. At different NaCl concentrations (2%, 4% and 6.5%), the growth ability was evaluated in the modified GM17 broth media. The change of the color as the evidence for cell growth (Nikita & Hemangi, 2012). Growth at pH 9.6, was detected by cultivating of isolates in the GM17 broth media with pH 9.6 (by pH adjustment with NaOH).

Arginine hydrolysis and gas production from citrate and Reduction of Methylene Blue
Inverted Durham tubes and Reddy broth were applied in this test. The color of cultures usingargininechanged from yellow to violet. To reduce Methylene Blue, overnight cultures were transferred into GM17 broth tubes with Methylene Blue.

Carbohydrate fermentations
The properties of the isolates were further determinedaccording to the profiles related to their sugar fermentation. Sixteen different sugars were utilized. In each test, the strains were inoculated in 3 ml of Phenol red base broth media containing 150 µl of filter sterilized (0,22 µm, Merck Millipore) 10% sugar solutions (0.5%). The acidification as a measure of fermentation ability is reflected in color change from red to yellow.

DNA extraction
To extract the DNA, the cultures of isolates grown in MRS and GM17 broth for 18h were employed. Genomic DNA extraction was donethrough phenolchloroform extraction, which is a modified bacterial DNA extraction protocol depicted by Federici et al.,and the samples of DNA were maintained at -20°C (Federici et al., 2014). A single colony of each isolate was inoculated into 10 ml of the appropriate medium broth and incubated overnight at 37°C. Using centrifugation at 7500-8000 rpm for 5 min, the cells were harvested, and genomic DNA was isolated based on a modified genomic DNA isolation protocol (Ashmaig, Hasan, & El Gaali, 2009). The collected bacterial pellet was resuspended in 2.5 ml of TE buffer (1M Tris-HCl, 0.5 mM EDTA pH 8.0) and washed two times with the same buffer. Once washed,the pellet was resuspended into 250 μl of lyses solution (1% (w/v) SDS, 0.2 mol NaOH). Through gentle shaking, the cell suspension was incubated for 1 h in a water bath at 65°C . The solution was gently emulsified by an equal amount of TE buffer saturated phenolchloroform (1:1); it was thencentrifuged for 5 min at 7500-8000 rpm, and the aqueous phase was transferred intonew tubes. Next, the aqueous solution waswashed two times with an equal amount of chloroform: isoamylalcohol, 24:1, and it was centrifugated for 5 min at 7500-8000 rpm. Oncethe aqueous phase was centrifuged, it was transferred to Eppendorf tube (400 μl/ tube) to which1/10 volume of three molar sodium acetate (pH 2.5) was added. Via adding 2 volumes of ice cooled absolute ethanol, the nucleic acids were precipitated. Subsequently, the mixture underwent centrifugationat 14000 rpm for 10 min; after that,the supernatant was discarded and the created pellet was washed two times by 70% ethanol. The remaining ethanol was eliminated through drying the pelletat room temperature. It was then dissolved in TE buffer and kept at -20°C.

Multiple sequence alignment
Ultimately, the amplified genes were obtained from the gels by use of a QIA quick gel extraction kit (Bioneer, USA) according to the the manufacturer's instructions. Buffer 1 (gel binding buffer) was added to the cutted piece of gel, incubated for 10 min at 60°C to completely desolve the gel. The solution was transferred to the DNA binding column tube, centrifuged for 1 min at 13000 rpm and poured in the flow-through, re-assembling the DNA binding filter column with 2.0 ml collection tube. 500 μl of buffer 2 (cell lysis buffer) was added to the DNA binding column tube, centrifuged for 1 min at 13000 rpm., poured in thethrough, re-assembling the DNA binding filter column with the 2.0 ml collection tube and dried via another centrifugation for 1 min at 13000 rpm. DNA was eluted from column by adding 30 μl buffer 3 (EB) to the center of the DNA binding filter column and remaining for at least 1 min at room temperature and centrifugation 1 min at 13000 rpm. The purified DNA fragments of amplified genes for 16S RNA were finally subjected to sequencing service (Macrogen, South Korea) for standard sequencing (Poormontaseri, Hosseinzadeh, & Shekarforoush, 2014).

DNA sequence Analysis
DNA sequences were obtained by use of forward and reverse primers assembled and edited through the use of BioEdit sequence alignment editor version 5.0.9. Using BLAST, the homology was searched in the Genbank DNA database, and the sequence similarity was estimated.

Lactic Acid Bacteria Isolation
From the collected samples, approximately 280 colonies were picked at random from selective media for each group of LAB.

Phenotypic Identification
Selected colonies from the fermented food samples showed comparable differences in their colony morphology (size, shape, shine and color). Among the 280 Gram-positive isolates that were extractedfrom the dairy products, 117 (all belonging to the LAB family) were negative for catalase and oxidase activity andnon-spore forming bacteria . A large number ofisolates (60 isolates) had a rod shape, therebypossibly related tothe genus Lactobacillus; the rest were cocci (57 isolates).

Physiological and Biochemical Identification
The results of identification of coccoid shaped isolates are shown in Table 1. According to obtained results, the strains were Streptococcus (29 isolates), enterococci (12 isolates) and lactococci (16 isolates). The ability of lactobacilli isolates to ferment different types of carbohydrate is shown in Table 2.

Figure 1
The percentage of LAB species isolated from collected samples In addition, we found that cocci especially those cultured by pouring plate, have complitely different forms, however with the aid of a microscope, they were seen as spherical or oval, mostly forming short or long chain and in single or pairs. Bacilli were observed to be so differently from each other under a microscope; although, on the plate, they were almost similar except L. bulgaricus with a star shape. Diversity of bacterial species can be related to different factors, such asraw milk composition and the breed of the animal,regional influences, the kind of the utilized culture medium, effect of reducing pH environment at different stages of growth, concentration of salt in the environment, and the existenceof calcium (Kirdar, 2012;Rahimi et al., 2012;Wright & Klaenhammer, 1983). In order to classify cocci shaped isolates, growth ability at 10°C, 40°C, and 45°C as well as growth at 2%, 4% and at the consentration of 6.5% NaCl were used. Streptococcus thermophilus was not able to show growth at 10°C, but it had a good growth at 40°C and 45°C; 47 isolates showed these characteristics. Enterococcus species could grow at 10°C, 40°C and 45°C; 3 isolates showed these characteristics. Unlike other species, Lactococcus species indicated growth at 10°C but had no growthat 45°C and only 26isolates possessed such property. L. lactis subsp. lactis strain could grows poorly at 45°C, while L. lactis subsp. cremoris grew just at 10°C. S. thermophilus and L. lactis subsp. cremoris did not have the ability to grow at salt concentrations of 4 and 6.5%, while L. lactis subsp. lactis and Enterococcus species could grow at different levels of salt. Reduction methylene blue, growth at a pH of 9.6, arginine hydrolysis, and citrate utilization and carbohydrate fermentations also were examined. Only 11 isolates were able to reduce methylene blue and hydrolyze the arginine. At a pH of 9.6, very few (only 2 isolates) could grow and 5 of them were able to produce gas from citrate. Cocci shape isolates capable of fermenting carbohydrates(raffinose, mannitol, ribose, lactose, maltose, galactose)were also evaluated. S. thermophilus and L. lactis subsp. cremoris bacteria had less ability to ferment carbohydrates, while Enterococcus species and some species of Lactococcus are able to ferment majority of tested sugars. S. thermophilus and L. lactis subsp. cremoris bacteria differ in the fermentation of galactose; S. thermophilus poorly ferments galactose because theits enzyme beta-galactosidase is not able to ferment this sugar. Similarly, most researchers agree on the fermentation of lactose by cocci bacteria (Begovic et al., 2011; Patil, Pal, Anand, & Ramana, 2010). On the contrary, atypical characteristic of some Streptococcus strains is their galactose fermentation. Almost none of LAB cocci were able to ferment raffinose, however, they are able to break the disaccharide lactose into glucose and galactose that can be used for energy. For identification of bacilli shaped isolates, carbon dioxide from glucose and carbohydrate fermentation tests were performed by applying 13 different type of carbohydrates. The biochemical tests showed that Lactobacillus species could be divided to three groups. Group I comprises obligately homofermentative strains (L. acidophilus, L. delbruckii, L. helveticus). The second category includes facultative heterofermentative strains (L. casei, L. plantarum), and group III are obligately heterofermentative strains (L. brevis, L .fermentum); This means that for the fermentation of sugar, only the 6-PG/PK pathway is accessible. In order to differentiate homofermentative and heterofermentative LAB, the ability of the isolates was studied in the fermentation of pentose sugars such as ribose and xylose. All cocci isolates were able to ferment pentose in order to identify species more accurately. This survey and similar studies indicate the approximate different sugars fermented by Lactobacillus species ( (Maqsood et al.,  2013). In a previously reported research by Kirdar (2012) in meaditerian area, three Lactobacillus species were noted. Unlike to other lactobacilli, L. casei subsp. pseudoplantarum is able to obtain energy and produce acid from most of the carbohydrates (Abdullah & Osman, 2010). The ability to ferment galactose is considered as the main factor for distinction between L. helveticus species as galactose positive and L. bulgaricus known as galactose negative. L. lactis and L. acidophilus ferment sugars in the same way, however, the only distinction between them is in the fermentation of raffinose, in which L. lactis is not able to use the sugar for its energy supply. Roshanzade et al. reported the similar action of these two species in the fermentation of sugars (RoushanZadeh, Eskandari, Shekarforoush, & Hosseini, 2014). Bacterial strains oxidize environmental energy sources by producing different enzymes; however, using energy sources depends on the synthesis of specific enzymes by each bacteria (Ashmaig et al.,  2009). According to the literature, new bacterial species identified are discovered in almost the same studies carried out previousely, pointing that the main reason for diverzification could be adaptation to the local conditions of the region. Biochemical tests for the representative LAB isolated from traditional yogurts in various parts of Fars province revealed the occurrence of 52 (44.44%) L. lactis subsp cremoris and 65 (55.56%) L. mesenteroides subsp cremoris among LAB cocci and regardingLAB bacilli ,L. helveticus 85 (15.3%); L. plantarum 124 (22.3%); L. brevis 117 (21%) ;L. casei subsp. casei 86 (15.5%) and L. delbruckii subsp. bulgaricus 144 (25.9%) were detected (Azadnia & Khan Nazer, 2009). To investigate different species isolated from Iran using phenotypical and molecular tests, Tajabadi Ebrahimi et al. performed a study on traditional Iranian dairy products. Isolated species belonged to L. plantarum (cheese, fermented milk, yogurt, kashk), L. brevis (cheese, yogurt), L. casei (cheese, yogurt, fermented milk), L. acidophilus (yogurt), L. jensenii (cheese), L. salivarius (yogurt), L. lactis (cheese), L. alimentarium (yogurt), L. rhamnosus (cheese), L. arieminis (

Genotypic identification
Since 1990, some alternatives have emerged for the classical phenotypic and biochemical identification of LAB . These methods might not be sufficient to definitively attribute a strain to a certain species. To thoroughly identify and classify the species in bacterial systematics, polyphasic approaches (Coeuret, Dubernet, Bernardeau, Gueguen, & Vernoux, 2003), such asphenotypic, chemotaxonomic, and genotyping methods are still recommended. To identify the LAB species more accurately, PCR assays were used with primers that target 16S rRNA gene (Table 3). To confirm the belonging to certain species, the nucleotide sequences of the 16S rRNA gene related to all the isolates were examined and specified using the BLAST program on NCBI. The results were categorized based onthe maximum identity andrecorded in terms of coverage. The sequence similarity with 90% or higher cut-off was regarded as significant.The optimum hit was considered as the sequence with the most maximum similarity to the query sequence. With the current rapid growth in biotechnology, more and more molecular techniques are utilizedin the genetic diversity studies on LAB. Typing methods based onmajor advantages because of their highly discriminatory power, being applicable universally and having independent culture. Methods like pulsed field gele electrophoresis (PFGE), DNA/DNA hybridization, ribotyping, polymerase chain reaction (PCR), real-time PCR hybridization and sequencing of rRNA are able to distinguish even the close related strains They made use of a set of Lb. reuteri and Lb. plantarum strains. These strains weresystematically characterized via phenotypic tests, DNA-DNA homology, REA, automated PCR sequencing of rRNA, ribotyping, and RAPD. Their work clearly elucidates that every method has its own upsides and downsides,and that one technique cannot bethe answer to all problems, but rather different methods complement one another.

CONCLUSION
In the present study, in-vitro approaches were chosen to examine the diversity of the LAB in dairy products from Iran. Previous studies have been conducted on LAB isolates from the Iranian traditional cheeses and yogurts, with less attention to others traditional and industrial dairy products. This work showed that other fermented dairy products could be significant sources of LAB. Some studies reflected the fact that LAB strains detected from dairy products, produce more pleasant flavor and volatile compounds than industrial strains. Local LABs are highly potential as starters or adjuncts. To optimally utilize novel strains, the specific conditions of use have to be definedand more detailed research must be conducted on their technological features. Tamime, A., & Marshall, V. (1997). Microbiology and technology of fermented milks.