The influence of starter cultures on the lactic acid bacteria microbiota of Petrovac sausage

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Bojana Milicevic
Vladimir Tomović
Bojana Danilović
Dragiša Savić


fermented sausages, lactic acid bacteria, Petrovac sausage, 16S rDNA sequencing


Petrovac sausage (Petrovská klobása) is a high-quality fermented dry sausage produced traditionally in the municipality of Ba?ki Petrovac (Vojvodina, Serbia). The product is characterised by specific and recognised texture, aroma and colour, produced without additives or preservatives. Lactic acid bacteria (LAB) microbiota plays an important role in production of the sausage. The aim of the paper is to monitor the changes in LAB during the production of Petrovac sausage. Samples of sausages were prepared without and with the addition of starter culture Staphylococcus xylosus as well as combined starter culture Lactiplantibacillus plantarum and S. xylosus, and produced at two different temperature ranges. A total number of 495 strains were isolated from 33 samples of Petrovac sausage during 120 days of production process. Characterisation of the isolates was performed by phenotypic tests, while molecular identification of the representative strains was done by 16S ribosomal DNA sequencing. The total number of LAB was about 8 log (Colony Forming Unit (CFU))/g in all samples, while the number of staphylococci was about 4 log CFU/g. Molecular identification confirmed that all isolates belonged to the following species: Levilactobacillus brevis, Leuconostoc mesenteroides, Lactiplantibacillus plantarum and Pediococcus pentosaceus. Lactobacilli and Leuconostoc spp. dominate the total LAB strains, while P. pentosaceus was isolated at the lowest frequency.


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Albano H., Todorov S.D., van Reenen C.A., Hogg T., Dicks L.M. and Teixeira P. 2007. Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from “Alheira,” a fermented sausage traditionally produced in Portugal. Int J Food Microbiol. 116(2):239–47. ijfoodmicro.2007.01.011
Albano H., Van Reenen C.A., Todorov S.D., Cruz D., Fraga L., Hogg T.,et al. 2009. Phenotypic and genetic heterogeneity of lac-tic acid bacteria isolated from ‘’Alheira,” a traditional fermented sausage produced in Portugal. Meat Sci. 82:387–398. https://doi. org/10.1016/j.meatsci.2009.02.009
Ambrosiadis J., Soultos N., Abrahim A. and Bloukas J.G. 2004. Physicochemical, microbiological and sensory attributes for the characterization of Greek traditional sausages. Meat Sci. 66:279–287.
Ammor M.S. and Mayo B. 2007. Selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: an Update. Meat Sci. 76:138–146. https://doi. org/10.1016/j.meatsci.2006.10.022
Anba-Mondoloni J., Champomier-Vergès M.C., Zagorec M., Leroy  S., Dordet-Frisoni E., Planchon S. and Talon R. “The Genetics of Microbial Starters,” in Handbook of Fermented Meat and Poultry: Second Edition, F. Toldrá, Ed., pp. 161–168, Wiley Blackwell, 2015.
Benito M., Serradilla M.J., Ruiz-Moyano S., Martin A., Pérez-Nevado F. and Cordoba G. 2008. Rapid differentiation of lactic acid bacteria from autochthonous fermentation of Iberian dry-fermented sausages. Meat Sci. 80:656–661. https://doi. org/10.1016/j.meatsci.2008.03.002
Bolumar T., Sanz Y., Flores M., Aristoy M.C., Toldra F. and Flores J. 2006. Sensory improvement of dry-fermented sausages by the addition of cell-free extracts from Debaryomyces hansenii and Lactobacillus sakei. Meat Sci. 72:457–466. https://doi. org/10.1016/j.meatsci.2005.08.010
Bonomo M.G., Ricciardi A., Zotta T., Parente E. and Salzano G. 2008. Molecular and technological characterization of lactic acid bacteria from traditional fermented sausages of Basilicata region (Southern Italy). Meat Sci. 80(4):1238–1248. https://doi. org/10.1016/j.meatsci.2008.05.032
Borovic B., Velebit B., Veskovic S., Lakicevic B. and Baltic T. 2017. The characterization of lactic acid bacteria isolated during the traditional production of Uzicka sausage. IOP Conference Series: Earth and Environmental Science,  59th International Meat Industry Conference MEATCON2017, Zlatibor, Serbia, Volume 85 (1), pp.0120179,1.10.2017–4.10.2017. https://doi. org/10.1088/1755-1315/85/1/012079
Casaburi A., Blaiotta G., Mauriello G. and Pepe O. 2005. Technological activities of Staphylococcus carnosus and Staphylococcus simulans strains isolated from fermented sausages. Meat Sci. 71(4):643–650. meatsci.2005.05.008
Casaburi A., Di Monacoa R., Cavellaa S., Toldrà F., Ercolini D. and Villani F. 2008. Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits. Food Microbiol. 25:335–347. fm.2007.10.006
Casquete R., Benito M., Martin A., Ruiz-Moyano S., Aranda E. and Córdoba M.G. 2012. Microbiological quality of salchichón and chorizo, traditional Iberian dry-fermented sausages from two different industries, inoculated with autochthonous starter cul-tures. Food Control. 24(2):191–198. foodcont.2011.09.026
Cocconcelli P.S. and Fontana C. 2008. “Characteristics and Applications of Microbial Starters in Meat Fermentations,” in Meat Biotechnology, F. Toldrá, Ed., pp. 129–148.
Comi G., Urso R., Iacumin I., Rantsiou K., Cattaneo P., Cantoni C., et  al. 2005. Characterization of naturally fermented sausages produced in the north east of Italy. Meat Sci. 69:381–392.
Danilovic B. 2012. Changes in the population of lactic acid bacteria during the production of petrovac sausage (Petrovská klobása). D. Sc. thesis, Faculty of Technology, University of Nis, Leskovac.
Danilovic B., Jokovic N., Petrovic Lj., Veljovic K., Tolinachki M. and Savic D. 2011. The characterisation of lactic acid bacteria during the fermentation of an artisan Serbian sausage (Petrovska Klobasa). Meat Sci. 88: 668–674. meatsci.2011.02.026
Danilovic B., Dzinic N., Milosavljevic N. and Savic D. 2018. Influence of processing conditions on the lactic acid bacteria population of a traditional sausage. Romanian Biotechnol Lett. 23(3):13661–13668.
El Adab S., Essid I. and Hassouna M. 2014. Effect of starter cultures on microbial and physicochemical parameters of a dry fermented poultry meat sausage. African J Biotechnol. 13(43):4155–4164.
Erdogrul O.T., Cetin O. and Ergun O. 2002. A study on metabolic and antimicrobial activities of Pediococcus pentosaceus isolated from fermented sausages. Pak J Biol Sci. 5(5):594–596. https://
Essid I. and Hassouna M. 2013. Effect of inoculation of selected Staphylococcus xylosus and Lactiplantibacillus plantarum strains on biochemical, microbiological and textural characteristics of a Tunisian dry fermented sausage. Food Control 32(2):707–714.
Fiorentini A.M., Sawitzki M.C., Bertol M., Cunha Junior A. and Sant’Anna E.S. 2010. Influence of a native strain of Staphylococcus xylosus on the microbiological, physicochem-ical and sensorial characteristics on milano salami type. Brazil Arch Biol Technol 53(4):961–974. S1516-89132010000400027
Hammes W., Haller D. and Ga¨nzle M. 2008. Fermented meat. In: Edward R. (Ted) Farnworth (ed.) Handbook of fermented func-tional foods, Taylor & Francis, Boca Raton, pp. 291–321. https://
Heinz G. and Hautzinger P. 2007, June 1. Meat processing technology. For small-to medium scale producers. Food and Agriculture Organization of the United Nations (FAO), Regional Office for Asia and the Pacific, Bangkok. ISBN: 978-974-7946-99-4.
Hosseini H. and Pilevar Z. 2017. Effects of starter cultures on the properties of meat products: a review. Ann Res Rev Biol. 17(6):1–17.
Ikonic P., Jokanovic M., Petrovic Lj., Tasic T., Skaljac S., Sojic B., et al. 2016. Effect of starter culture addition and processing method on proteolysis and texture profile of traditional dry-fermented sausage Petrovská klobása. Int J Food Prop. 19:1924– 1937.
Ikonic P., Jokanovic M., Tasic T., Skaljac S., Sojic B., Tomovic  V., et al. 2015. The effect of different ripening conditions on proteolysis and texture of dry-fermented sausage Petrovská klobása. Procedia Food Sci. 5:97–100. profoo.2015.09.026
Johansson G., Berdague J.L., Larsson M., Tran N. and Borch E. 1994. Lipolysis, proteolysis and formation of volatile components during ripening of fermented sausage with Pediococcus pento-saceus and Staphylococcus xylosus as starter culture. Meat Sci. 38:203–218.
Jokanovic M., Ikonic P., Skaljac S., Tasic T., Tomovic V., Sojic B., et al 2017. Proteolysis and texture profile of traditional dry-fermented sausage as affected by primary processing method. Meat Technol. 58: 103–109.
Jokanovic M., Petrovic Lj., Ikonic P., Tomovic V., Dzinic N., Savatic  S., et al 2010. Sensory properties of Petrovská klobása (dry-fermented sausage) ripened in traditional and industrial conditions. J Proc Energy Agricul. 14:153.
Kovacevic D., Mastanjevic K., Subaric D., Jerkovic I. and Marijanovic Z. 2010. Physico-chemical, colour and textural properties of Croatian traditional dry sausage (Slavonian kulen). Meat 12:270–276.
Kozachinski L., Drosinos E., Chaklovica F., Cocolin L., Gasparik-Reichardt J. and Veskovic S. 2008. Investigation of microbial association of traditionally fermented sausages. Food Technol Biotechnol. 46:93–106.
Kumar P., Chatli M.K. and Verma A.K. 2017. Quality, functionality, and shelf life of fermented meat and meat products: a review. Crit Rev Food Sci. and Nutr. 57:2844–2856. 080/10408398.2015.1074533
Laranjo M., Elias M. and Fraqueza M.J. 2017. The use of starter cultures in traditional meat products. J Food Quality 2017:1–18.
Lee J.Y., Kim C.J. and Kunz B. 2006. Identification of lactic acid bac-teria isolated from kimchi and studies on their suitability for application as starter culture in the production of fermented sausages. Meat Sci. 72(3):437 445. meatsci.2005.08.013
Leistner L. 1995. Stable and safe fermented sausages worldwide. In: Campbell-Platt G. and Cook P.E. (eds.), Fermented meats. Blackie Academic & Professional, Glasgow, Scotland, pp. 160– 175.
Leroy F., Verluyten J. and De Vuyst L. 2006. Functional meat starter cultures for improved sausage fermentation. Int J Food Microbiol. 106(3):270–285. ijfoodmicro.2005.06.027
Lizaso G., Chasco J. and Beriain J. 1999. Microbiological and bio-chemical changes during ripening of salcichon, a Spanish dry cured sausage. Food Microbiol. 16:219–228. https://doi. org/10.1006/fmic.1998.0238
Martinovic A. and Veskovic-Moracanin S. 2006. Application of starter cultures in the meat industry. Meat Technol. 47(5–6):216–230.
Najjari A., Boumaiza M., Jaballah S., Boudabous A. and Ouzari H. 2020. Application of isolated Lactobacillus sakei and Staphylococcus xylosus strains as a probiotic starter culture during the industrial manufacture of Tunisian dry-fermented sausages. Food Sci. Nutr. 8:4172–4184. fsn3.1711
Nieto-Lozano J.C., Reguera-Useros J.I., Peláez-Martínez M.D.C., Sacristán-Pérez-Minayo G., Gutiérrez-Fernández A.J. and Torre A.H.D. 2010. The effect of the pediocin PA-1 produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens in Spanish dry-fermented sausages and frankfurters. Food Control. 21:679–685. https://doi. org/10.1016/j.foodcont.2009.10.007
Nikolic M., Terzic-Vidojevic A., Jovcic B., Begovic J., Golic N. and Topisirovic L. 2008. Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat’s milk cheese. Int J Food Microbiol. 122:162–170. ijfoodmicro.2007.11.075
Paik H.D. and Lee J.Y. 2014. Investigation of reduction and tolerance capability of lactic acid bacteria isolated from kimchi against nitrate and nitrite in fermented sausage condition. Meat Sci. 97:609–614.
Papamanoli E., Tzanetakis N., Litopoulou-Tzanetaki E. and Kotzekidou P. 2003. Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties. Meat Sci. 65(2):859–867.
Petrovic Lj., Dzinic N., Tomovic V., Ikonic P. and Tasic T. 2007. Code of practice – registered geographical indication Petrovská klobása. Decision No. 9652/06 G-03/06, Intellectual Property Office. Republic of Serbia.
Pidcock K., Heard G.M. and Henriksson A. 2002. Application of nontraditional meat starter cultures in production of Hungarian salami. Int J Food Microbiol. 76:75–81. S0168-1605(02)00002-8
Rantsiou K. and Cocolin L. 2006. New developments in the study of the microbiota of naturally fermented sausages as determined by molecular methods: a review. Int J Food Microbiol. 108:255– 267.
Semjonovs P. and Zikmanis P. 2008. Evaluation of novel lactose-positive and exopolysaccharide-producing strain of Pediococcus pentosaceus for fermented foods. Eur Food Res Technol. 227(3):851–856.
Skoci?ska K., Wójciak K. and Zielis?ka D. 2016. Probiotic microorganisms in dry fermented meat products. In: Rao V. and Rao L.G.(eds.), Probiotics and prebiotics in human nutrition and health. InTech., Croatia, pp. 279–300.
Søndergaard A.K. and Stahnke L.H. 2002. Growth and aroma production by Staphylococcus xylosus, S. carnosus and S. equorum – a comparative study in model systems. Int J Food Technol. 75(1–2):99–109.–2
Stiles M.E. 1994. Bacteriocins produced by Leuconostoc species. J Diary Sci. 77(9):2718–2724. S0022-0302(94)77214-3
Toldra F. 2002. Dry-cured meat products. Food & Nutrition Press, Trumbull, CT. pdf/10.1002/9780470385111.fmatter
Tosukhowong A., Visessanguan W., Pumpuang L., Tepkasikul P., Panya A. and Valyasevi R. 2011. Biogenic amine formation in Nham, a Thai fermented sausage, and the reduction by commercial starter culture, Lactiplantibacillus plantarum BCC 9546. Food Chem. 129(3):846–853. foodchem.2011.05.033
Urso R., Comi G. and Cocolin L. 2006. Ecology of lactic acid bacteria in Italian fermented sausages: isolation, identification and molecular characterization. System Appl Microbiol. 29(8):671– 680.
Vasilev A., Aleksic B., Tarbuk A., Dimitrijevic M., Karabasil N., Cobanovic N., et al. 2015. Identification of lactic acid bacteria isolated from Serbian traditional fermented sausages Sremski and Lemeski kulen. Procedia Food Sci. 5:300–303. https://doi. org/10.1016/j.profoo.2015.09.071
Versalovic J., Schneider M., de Brujin F. and Lupski J.R. 1994. Genomic fingerprinting of bacteria using repetitive sequence based PCR (rep-PCR). Methods Mol Biol. 5:25–40.
Veskovic-Moracanin S. 2010. Lactic acid bacteria bacteriocins as natural food protectors: possibilities of application in meat industry. Meat Technol. 51(1):83–94.
Zdolec N., Hadziosmanovic M., Kozacinski L., Cvrtila Z., Filipovic I., Skrivanko M. and Leskovar K. 2008. Microbial and physico-chemical succession in fermented sausages produced with bacteriocinogenic culture of Lactobacillus sakei and semi-purified bacteriocin mesenterocin Y. Meat Sci. 80:480–487. https://doi. org/10.1016/j.meatsci.2008.01.012