Genetic polymorphism of lactic acid bacteria isolated from “Pirot ‘ironed’ sausage” from Serbia
Keywords:Pirot ‘ironed’ sausage, GTG5 PCR, Lactobacillus sakei, Leuconostoc mesenteroides
- Diversity of lactic acid bacteria (LAB) in six types of traditional “Pirot ‘ironed’ sausage” was determined.
- The morphological, biochemical and molecular characteristics of the isolates were tested.
- GTG5 PCR was determined as the best discriminatory technique for fast LAB screening.
- Lactobacillus sakei and Leuconostoc mesenteroides were the dominant species.
- High genetic heterogeneity of L. sakei isolates could be important for the specific nature of the sausage.
Abstract: The “Pirot ‘ironed’ sausage” (Pis) is a traditional, dry, fermented product originating from the town of Pirot, situated in southeastern Serbia. This product is made from different types of meat (goat, sheep, beef and donkey) and spices, without additives or starter cultures. Pis is an organic, unprocessed product, without heat or smoke treatment. The aim of this study was to characterize 120 isolates of lactic acid bacteria (LAB) from Pis produced by six different brands during a two-year period using phenotypic and genetic identification. Preliminary characterization of the LAB was based on general morphology and biochemical tests. Repetitive elements such as REP, BOX and GTG5, found in the genome of these bacteria, and randomly amplified polymorphic DNA-polymerase chain reaction (RAPD PCR) sequences were used for determination of genetic polymorphism. Identification by 16S rRNA gene sequencing showed the presence of only two LAB species, Lactobacillus sakei (76%) and Leuconostoc mesenteroides (24%). However, genetic polymorphism was detected using fingerprinting methods. In comparison with other primers, the profiles obtained with GTG5 showed the highest heterogeneity for most of the tested isolates, with sequencing results additionally confirming its discriminatory power. In addition, M13 RAPD primer also produced satisfactory separation of the tested isolates.
Received: September 20, 2018; Revised: October 31, 2018; Accepted: November 5, 2018; Published online: November 6, 2018; Published online: November 9, 2018
How to cite this article: Bogdanović S, Jelušić A, Berić T, Nikolić I, Danilović B, Stanković S, Dimkić I. Genetic polymorphism of lactic acid bacteria isolated from “Pirot ‘ironed’ sausage” from Serbia. Arch Biol Sci. 2019;71(1):95-102.
Papamanoli E, Tzanetakis N, Litopoulou-Tzanetaki E, Kotzekidou P. Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties. Meat Sci. 2003;65(2):859-67.
Danilović B, Joković N, Petrović L, Veljović K, Tolinački M, Savić D. The characterisation of lactic acid bacteria during the fermentation of an artisan Serbian sausage (Petrovská Klobása). Meat Sci. 2011;88(4):668-74.
Hugas M, Monfort JM. Bacterial starter cultures for meat fermentation. Food Chem. 1997;59(4):547-54.
Urso R, Comi G, Cocolin L. Ecology of lactic acid bacteria in Italian fermented sausages: isolation, identification and molecular characterization. Syst Appl Microbiol. 2006;29(8):671-80.
Riboulet-Bisson E, Sturme MH, Jeffery IB, O'Donnell MM, Neville BA, Forde BM, Lawlor PG. Effect of Lactobacillus salivarius bacteriocin Abp118 on the mouse and pig intestinal microbiota. PLoS One. 2012;7(2):e31113.
Adiguzel CG, Atasever M. Phenotypic and genotypic characterization of lactic acid bacteria isolated from Turkish dry fermented sausage. Rom Biotechnol Lett. 2009;14(1):4130-8.
Rantsiou K, Cocolin L. New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods: a review. Int J Food Microbiol. 2006;108(2):255-67.
Tran KT, May BK, Smooker PM, Van TT, Coloe PJ. Distribution and genetic diversity of lactic acid bacteria from traditional fermented sausage. Food Res Int. 2011;44(1):338-44.
Gancheva A, Pot B, Vanhonacker K, Hoste B, Kersters K. A polyphasic approach towards the identification of strains belonging to Lactobacillus acidophilus and related species. Syst Appl Microbiol. 1999; 22(4):573-85.
Gevers D, Huys G, Swings J. Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. FEMS Microbiol Lett. 2001;205(1):31-6.
Rantsiou K, Drosinos EH, Gialitaki M, Metaxopoulos I, Comi G, Cocolin L. Use of molecular tools to characterize Lactobacillus spp. isolated from Greek traditional fermented sausages. Int J Food Microbiol. 2006;112(3):215-22.
Van Hoorde K, Vandamme P, Huys G. Molecular identification and typing of lactic acid bacteria associated with the production of two artisanal raw milk cheeses. Dairy Sci Technol. 2008;88(4-5):445-55.
Mohammed M, El-Aziz HA, Omran N, Anwar S, Awad S, El-Soda M. Rep-PCR characterization and biochemical selection of lactic acid bacteria isolated from the Delta area of Egypt. Int J Food Microbiol. 2009;128(3):417-23.
Greco M, Mazzette R, De Santis EPL, Corona A, Cosseddu AM. Evolution and identification of lactic acid bacteria isolated during the ripening of Sardinian sausages. Meat Sci. 2005;69(4):733-9.
Dimkić I, Stanković S, Nišavić M, Petković M, Ristivojević P, Fira D, Berić, T. The profile and antimicrobial activity of Bacillus lipopeptide extracts of five potential biocontrol strains. Front Microbiol. 2017;8:925.
Bonomo MG, Ricciardi A, Zotta T, Parente E, Salzano G. Molecular and technological characterization of lactic acid bacteria from traditional fermented sausages of Basilicata region (Southern Italy). Meat Sci. 2008;80(4):1238-48.
Pavel AB, Vasile CI. PyElph-a software tool for gel images analysis and phylogenetics. BMC bioinformatics. 2012;13(1):9.
Čolo J, Mihajlović S, Tolinački M, Alkić M, Popović D, Kojić M, Terzić-Vidojević A. Characterization of lactic acid bacteria isolated from Bosnian artisanal dry fermented sausage (sudžuk) during fermentation. Genetika. 2015;47(3):819-32.
De Vuyst L, Camu N, De Winter T, Vandemeulebroecke K, Van de Perre V, Vancanneyt M, Cleenwerck I. Validation of the (GTG)5-rep-PCR fingerprinting technique for rapid classification and identification of acetic acid bacteria, with a focus on isolates from Ghanaian fermented cocoa beans. Int J Food Microbiol. 2008;125(1):79-90.
De Urazza PJ, Gomez-Zavaglia A, Lozano ME, Romanowski V, De Antoni G. DNA fingerprinting of thermophilic lactic acid bacteria using repetitive sequence-based polymerase chain reaction. J Dairy Res. 2000;67(63):381-92.
Lanoot B, Vancanneyt M, Dawyndt P, Cnockaert M, Zhang J, Huang Y, Swings J. BOX-PCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus. Syst Appl Microbiol. 2004;27(1):84-92.
Wieser M, Busse HJ. Rapid identification of Staphylococcus epidermidis. Int J Syst Evol Microbiol. 2000;50(3):1087-93.
Stephenson DP, Moore RJ, Allison GE. Comparison and utilization of repetitive-element PCR techniques for typing Lactobacillus isolates from the chicken gastrointestinal tract. Appl Environ Microbiol. 2009;75(21):6764-76.
Hyytiä-Trees E, Lyhs U, Korkeala H, Björkroth J. Characterisation of ropy slime-producing Lactobacillus sakei using repetitive element sequence-based PCR. Int J Food Microbiol. 1999;50(3):215-9.
Cocolin L, Rantsiou K, Iacumin L, Urso R, Cantoni C, Comi G. Study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods. Appl Environ Microbiol. 2004;70(4):1883-94.
Comi G, Urso R, Iacumin L, Rantsiou K, Cattaneo P, Cantoni C, Cocolin L. Characterization of naturally fermented sausages produced in the North East of Italy. Meat Sci. 2005;69(3):381-92.
Rantsiou K, Drosinos EH, Gialitaki M, Urso R, Krommer J, Gasparik-Reichardt J, Cocolin L. Molecular characterization of Lactobacillus species isolated from naturally fermented sausages produced in Greece, Hungary and Italy. Food Microbiol. 2005;22(1):19-28.
Ammor MS, Mayo B. Selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: An update. Meat Sci. 2007;76(1):138-46.
Lee JY, Kim CJ, Kunz B. Identification of lactic acid bacteria isolated from kimchi and studies on their suitability for application as starter culture in the production of fermented sausages. Meat Sci. 2006;72(3):437-45.
Santos EM, Gonzalez-Fernandez C, Jaime I, Rovira J. Comparative study of lactic acid bacteria house flora isolated in different varieties of 'chorizo'. Int J Food Microbiol. 1999;39(1):123-8.
How to Cite
Authors grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution 4.0 International License that allows others to share the work with an acknowledgment of the work’s authorship and initial publication in this journal.