1 ANTIMICROBIAL ACTIVITY OF PEDIOCOCCUS STRAINS AGAINST SOME LISTERIA SPP. DURING CO- CULTIVATION UNDER STATIC GROWTH CONDITIONS Yulian Tumbarski* 1 , Tanya Pavlova 1 , Velichka Yanakieva 1 , Bogdan Goranov 1 , Rositsa Denkova-Kostova 2 Address(es): Assoc. Prof. Yulian Tumbarski, DVM, PhD. 1 University of Food Technologies, Technological Faculty, Department of Microbiology, 26, Maritsa Blvd., 4002 Plovdiv, Bulgaria. 2 University of Food Technologies, Technological Faculty, Department of Biochemistry and Molecular Biology, 26, Maritsa Blvd., 4002 Plovdiv, Bulgaria. *Corresponding author: tumbarski@abv.bg ABSTRACT Keywords: Antimicrobial activity, co-cultivation, Pediococcus sp., Listeria sp., pediocins INTRODUCTION The genus Pediococcus unifies Gram-positive, catalase-negative, oxidase- negative, facultatively aerobic, homofermentative coccus-shaped bacteria, which produce lactic acid as major end product of glucose fermentation (Franz et al., 2014). They are widely used as starter cultures in the fermentation of meat products, vegetables, sourdough bread making (Plessas et al., 2020) and silage production (Cai et al., 1999). Some Pediococcus strains have been isolated from dairy products (farm-style cheeses and Cheddar cheese) as a part of non-starter lactic acid bacteria (Gurira & Buys, 2005) or can be considered as spoilage bacteria in beer (Sakamoto & Konings, 2003; Iijima et al., 2007), wine (Beneduce et al., 2004) and other beverages. The members of genus Pediococcus are known to possess many health benefits such as protection against cardiovascular diseases, inhibition of pathogenic microorganisms in the gastrointestinal tract, and immunomodulatory effects that make them suitable for use as probiotic strains (Nghe & Nguyen, 2014). One of the most important features of Pediococcus spp. is their antimicrobial activity against broad range of foodborne pathogens and spoilage microorganisms. The inhibitory effect of pediococci is related to the production of lactic acid that reduces the pH level in the food matrix during fermentation process and the synthesis of antimicrobial peptides known as bacteriocins or pediocins, which find practical application in food biopreservation. According to the bacteriocin classification, pediocins belong to class IIa bacteriocins characterized as small unmodified peptides (<5 kDa) consisting of 36-48 amino acid residues (Porto et al., 2017). P. acidilactici and P. pentosaceus are among the main producers of pediocins and their isoforms with possible utilization in the food industry as biopreservatives prolonging the shelf life of different food products (Mora et al., 2003). Many studies revealed that pediocins represent effective antilisterial bacteriocins, which act specifically against the foodborne pathogen Listeria monocytogenes. In addition to their high antimicrobial potential, pediocins display some advantages (higher antilisterial activity, wide pH range effectiveness and heat stability) than nisin, a bacteriocin produced by Lactococcus lactis subsp. lactis, officially approved in many countries as generally recognized as safe (GRAS) and widely used as biopreservative in the food industry, especially in dairy sector (Jamuna & Jeevaratnam, 2004; Pal et al., 2010). Some studies reported that Pediococcus bacteriocins possess greater biopreservation properties in comparison with the conventional chemical preservatives such as sodium sulphite and sodium benzoate, which can cause various negative effects on human health (Skariyachan & Govindarajan, 2019). The lactic acid bacteria have always been of paramount importance in the food industry due to their application as starter cultures, probiotics and functional food ingredients, texture, flavour and aroma enhancers, and at last, but not at least – as biopreservation agents. In recent years, the use of Pediococcus spp. in food industry attracted the research attention due to their valuable technological properties, which are essential for the final food quality (Adesulu-Dahunsi et al., 2021). The bacteriocin production and high antimicrobial activity of Pediococcus spp. against some crucial foodborne pathogens such as L. monocytogenes is a significant prerequisite for their application as alternative increasing the safety of food products. Although the in situ antilisterial potential of Pediococcus spp. and its use in controlling growth of L. monocytogenes in meat products (Nieto-Lozano et al., 2010; Kingcha et al., 2012; De Azevedo et al., 2020) and milk (Somkuti & Steinberg, 2009) is well studied, little scientific information is available on the antimicrobial activity against other members of genus Listeria as well as on the kinetics of the antimicrobial effect on these pathogens during in vitro co- cultivation. Therefore, this research aimed to investigate the antimicrobial activity of three Pediococcus strains (P. pentosaceus, P. acidilactici and P. damnosus) against three Listeria spp. (L. monocytogenes, L. innocua and L. ivanovii) during co-cultivation under static growth conditions, and to determine the growth characteristics and basic kinetic parameters that could facilitate the selection of appropriate Pediococcus strains for production of safe fermented foods. MATERIAL AND METHODS Microorganisms Three Pediococcus strains (P. pentosaceus ATCC 33316, P. acidilactici NBIMCC 1603 and P. damnosus ATCC 29358) and three Listeria strains (L. monocytogenes NBIMCC 8632, L. innocua ATCC 33090 and L. ivanovii ATCC 19119) from the collection of the Department of Microbiology at the University of Food Technologies - Plovdiv, Bulgaria, were used in the experiments. Pediococcus strains were cultured in MRS broth, while Listeria strains were cultured on BHI agar at 37 °C for 24 h, and then stored at 4°C until use. Before the experiment, the strains were activated by propagation in MRS broth and BHI broth, respectively, and incubation at 37°C for 24 h. Pediococci are lactic acid bacteria widely used as starter cultures in the food industry, which produce lactic acid and bacteriocins (pediocins) possessing significant antilisterial potential. This research aimed to investigate the antimicrobial activity of three Pediococcus strains (Pediococcus pentosaceus, Pediococcus acidilactici and Pediococcus damnosus) against three Listeria strains (Listeria monocytogenes, Listeria innocua and Listeria ivanovii) to determine the growth characteristics and kinetic parameters during the process of co-cultivation at 37 ± 1°C. The results demonstrated that P. pentosaceus possessed a bacteriostatic effect on L. monocytogenes, but a bactericidal effect on L. innocua and L. ivanovii, and on the 60-th h no viable cells of both pathogens were detected. P. acidilactici demonstrated a strong bactericidal effect on the three Listeria strains, as L. monocytogenes was completely inhibited on the 48-th h and L. innocua and L. ivanovii were inhibited on the 60-th h of co-cultivation. In contrast, P. damnosus showed only a bacteriostatic effect on the three pathogens tested. Therefore, the strains P. pentosaceus and P. acidilactici can be successfully applied in the food industry as biopreservatives and as means to improve the food safety by inhibition of Listeria spp., which are among the most common pathogens in food products of animal origin. ARTICLE INFO Received 20. 8. 2022 Revised 7. 11. 2022 Accepted 15. 11. 2022 Published xx.xx.201x Regular article https://doi.org/10.55251/jmbfs.9360