Bacteriocin Producing Bacteria isolated from turkish Traditional Sausage Samples

In this study, traditional sausage samples from different provinces of Turkey (Gaziantep, Antalya, erzurum and Kahramanmaras) were obtained and one hundred three isolates were collected. Using the (GTG)5-PCR genomic fingerprint analysis method, seven of them were observed to be different and conventional tests of these isolates were performed. Molecular identification of two isolates carrying the bacteriocin gene and having antimicrobial activity by agar disc diffusion method was performed by 16S rRNA sequence analysis. As a result, the seven isolates were identified as Aerococcus urinaeequi (EK1), Streptococcus salivarius (EK2), Leuconostoc mesenteroides (EK3), Macrococcus caseolyticus (EK4), Lactococcus garvieae (EK5), Staphylococcus saprophyticus (EK6) and Lactobacillus sakei (EK7). Among these strains, it has been determined that Ln. mesenteroides and L. sakei carried the mecentericin and sacacin genes. When antimicrobial activity against different strains was examined, inhibition formations of Ln. mesenteroides and L. sakei on Enterococcus faecalis, Shigella dysenteriae and Escherichia coli O157: H7 were observed.

III bacteriocins. Helveticin M has recently been characterized and found to disrupt the cell wall of Gram-positive bacteria and the outer membrane of Gram-negative bacteria. In addition, it has been stated that class III bacteriocin is effective against both Gram-positive and Gram-negative bacteria 10 . Class IV bacteriocins contain spherical, heatsensitive, helix and post-translationally modified proteins consisting of 35-70 amino acids 9 .
Genes related to bacteriocin biosynthesis are usually found collectively and are encoded in plasmids, chromosomes or transposons. Bacteriocins are synthesized as biologically inactive prepeptides, usually containing a N-terminal leader peptide bound to the C-terminal propeptide 11,12 .
Bacteriocin-producing organisms are resistant to the bacteriocins they produce because they have specific immune proteins. Two separate bacteriological immune systems have been identified in the bacteriocin-producing cell which would be a special ABC carrier system consisting of immune protein and two or three subunits. These two immune systems work together to protect the bacteriocin-producing cells from their bacteriocin 5 .
The aim of the study was isolation and identification of lactic acid bacteria from traditional sausage samples collected from different locations of Turkey. Then, determination of whether the bacteria have bacteriocin gene or not and detection of antimicrobial effect of these isolates were aimed.

Isolation and molecular characterization of lactic acid bacteria
Lactic acid bacteria (LAB) were isolated from Turkish traditional sausages. The samples were collected from different provinces of Turkey; Gaziantep, Kayseri, Erzurum and Kahramanmaras. Each sample (25 gr) was mixed with 225 mL physiological water (0.9% NaCl) and homogenized in stomacher. Samples were diluted (10 -1 -10 -7 ) and each dilution (100 µL) were spread onto MRS and M17 agar plates and incubated at 37°C for 48 h. First of all, the growing colonies were spread on M17 and MRS agar to obtain pure cultures and were eliminated by morphology differences on plates. The pure, single and different colonies were stored in the Tryptic Soy Broth with 15% glycerol content at -86°C for further studies.
The total genomic DNA of each test strain was isolated according to the procedure of Promega™ Wizard® Genomic DNA Purification Kit. Afterwards, test isolates were subjected to rep-PCR [(GTG) 5 -PCR] with the special primer of (GTG) 5 elements to obtain genomic fingerprinting. The 16S rRNA gene regions of the isolates which would be considered to be different according to the rep-PCR analysis were amplified with universal primers by PCR and were subjected to the cloning procedure [13][14][15][16][17] .

Conventional identification
The isolates which were selected according to genomic fingerprinting were subjected to conventional tests for pH, temperature and salt (NaCl) requirements in growth media. The pH, temperature and NaCl requirements for bacterial growth were measured in M17 and MRS broth by following optical density at 600 nm wavelength with spectrophotometer by the method of Prescott et al. 18. Firstly, M17 and MRS broth media were prepared and, then a full loop of the test isolate on M17 and MRS agar was transferred to broth media and incubated at different temperatures (10-45°C) for 48 h. To measure the response to pH changes during growth, M17 and MRS broth media were prepared and pH of broth media was adjusted to different points at pH 3-11 range, before autoclaving. Then, a full loop of the test isolate on M17 and MRS agar was transferred to broth media and incubated at 37°C for 48 h. The salt (NaCl) requirement for growth was also tested in M17 and MRS broth medium containing 2-12 (w/v). For this, M17 and MRS broth media were prepared and salt at determined concentrations were added to broth media, before autoclaving. Then, a full loop of the test isolate on M17 and MRS agar was transferred to broth media and incubated at 37°C for 48 h 19 .
Colony morphology, Gram staining, motility, the presence of catalase and oxidase reactions and gas productions were also investigated 18,20,21 .

Determination of bacteriocin genes
The selected test isolates were evaluated according to bacteriocin genes with multiplex PCR reactions. 75 ng of purified DNA (2 µL) was used as the template in 30 µL total reaction mixture. Twenty-eight microliters of the reaction cocktail were prepared as follows: PCR Buffer (10X) 3 µL, MgCl 2 1.2 µL (25mM), dNTPs (10 mM) 0.6 µL, bovine serum albumin 1.2 µL (20 mg/mL), primers (5 µM) 1 µL, Taq polymerase (250 U) 0.3 µL and water 13.7 µL. Primers were given in Table 1. A negative control (no DNA) was included in each PCR assay. PCR reactions were performed with a 96-well ProFlexTM PCR System, using the following conditions: an initial denaturation at 95°C for 5 min; 30 cycles consisting of 94°C for 0.5 min, annealing at 55°C for 1 min, extension at 72°C for 2 min and a final polymerization at 72°C for 5 min before cooling at 4°C 22 .
For the multiplex-PCR reactions, products (2 µL) were mixed with 1.0 µL gel loading buffer (6X) and subjected to agarose (1% w/v) gel electrophoresis in Tris-Acetate-EDTA (TAE) buffer at 90 V and for 120 min. After separation, the fragments were stained with ethidium bromide solution (2 ml Etbr/100 ml 1X TAE buffer). The amplified DNA product was monitored using the Quantum Vilber Lourmat Gel Documentation System (Australia).

Agar disk diffusion method
In the agar disc diffusion test, pathogenic strains (Enterococcus faecalis ATCC 29212, Escherichia coli O157:H7 ATCC 35150, Shigella  Then, these discs were placed on the petri dishes where pathogenic bacteria were inoculated and incubated at 37°C for 24 h. At the end of the period, the petri dishes were removed to check if the inhibition zone was formed and the diameter of the zone was measured 23,24 .

ResUlts AND DisCUssiON
Traditional sausage samples from different locations of Turkey were collected. Isolation of lactic acid bacteria were carried out and one hundred three isolates were selected according to colony differentiation on petri plates. (GTG) 5 -PCR was performed to show distinction at species level (Fig.1) and according to differences, seven different isolates were decided to determine with 16S rRNA sequencing.
16S rRNA sequences of the seven isolates were amplified and cloned into E. coli JM101 strain. The obtained sequence was compared those in GenBank and EzTaxon. As given in Table 2; EK1, EK2, EK3, EK4, EK5, EK6 and EK7 were similar to Aerococcus urinaeequi, Streptococcus salivarius, Macrococcus caseolyticus, Leuconostoc mesenteroides, Lactococcus garvieae, Staphylococcus saprophyticus and Lactobacillus sakei, respectively. Also, phylogenetic analysis by using neighbour-joining method and data from 16S rRNA sequencing was carried out and given in Fig.2. As a result of the 16S rRNA gene sequences of the isolates, very close similarity rates (>98%) were obtained, as specified before in different experiments. Madigan and Martinko 25 reported that isolates having sequence similarity over 97% might belong to the same species.
These isolates were selected according to genetic polymorphism of (GTG) 5 region. Svec et al. 26 reported that (GTG) 5 -PCR was highly effective in the identification of lactobacilli isolated from food samples, it enabled the rapid and reliable identification of lactobacilli and other lactic acid bacteria, which were important in the food fermentation industries.
Karani et al. 27 revealed that M. caseolyticus strain developed in the presence of salt as high as 8%. Diaz et al. 15 stated that halophilic or halo-tolerance microorganisms tolerated high salt concentrations and this was making them very important in remediation. Considering this information, it was concluded that M. caseolyticus, which was not normally LAB, could be widely used in biotechnological processes because of maintaining vital functions even in high salt concentrations. Ramirez-Chavarin et al. 28 reported that thermotolerant lactic acid bacteria became dominant flora in foods cooked like sausage and act as bioprotective agents. Varsha and Nampoothiri 29 found in their study that the bacteria L. garvieae showed growth at 42°C. The optimum pH range of S. saprophyticus was 5-6.8 30 and Ln. mesenteroides' optimum pH was 5.5 31 .
Optimum pH range was 8.5-9 for A. urinaeequi 32 , 6-7 for S. salivarius 33 , 6 for L. sakei 34 and 9.6 for L. garvieae 35 . Karani et al. 27 found that the optimum pH range for M. caseolyticus was 6-11. Dhakar and Pandey 36 emphasized that bacteria, archaea and eukaryotic organisms that had the potential to develop in a wide pH range could be used in industrially important bioprocesses.

Determination of bacteriocin presence
The potentials of isolates to produce bacteriocin were analyzed using specific PCR. Mesentericin and sacacin genes were determined in two isolates, EK4 (Ln. mesenteroides subsp. dextranicum) and EK7 (L. sakei subsp. sakei) (data not shown), respectively.
Todorov and Dicks 37 were reported presence of mesentericin in Ln. mesenteroides subsp. dextranicum ST99 from Boza samples. de Paula et al. 38 were isolated Ln. mesenteroides SJRP55 from cheese samples and purified bacteriocins which were identical to mesentericin Y105 and mesentericin B105. Hechard et al. 39 purified and characterized mesentericin Y105 from L. mesenteroides.
Sawa et al. 40 isolated L. sakei D98 from rice malt and purified three novel bacteriocin belonging to class IIa and class IId. Moretro et al. 41 optimized sacacin P production from L. sakei CCUG 42687 in a completely defined medium. Jiang et al. 42 defined a novel sacacin from L. sakei LSJ61.

Antimicrobial effect of LAB on pathogenic strains
When the effects of the test isolates against different pathogenic microorganisms were investigated by agar disc diffusion method (

CONClUsiON
Reliable foods would be crucial for human growth and development. In order for food to be reliable, it is necessary to avoid process applications and to use natural additives. Bacteriocins, one of these natural additives, play an important role in food preservation by inhibiting both pathogenic and spoilage microorganisms. In this study, traditional sausage samples demonstrated a diverse of microorganisms and introduced bacteriocin production ability. It could be concluded that fermented foods had a good potential for bacteriocin producing microorganisms and these bacteriocins would be a decent source for food hygiene problems. In addition, (GTG) 5 -PCR, which is one of the rep-PCR methods, exhibited the distinction between foodborne test strains at species and subspecies level.

CONFliCt OF iNteRest
The authors declare that there is no conflict of interest.