Technological properties and safety evaluation of bacteriocinogenic Enterococcus strains isolated from raw meat and traditional meat products in Türkiye

Burak Geniş; Yasin Tuncer

doi:10.1186/s12866-026-05001-y

. 2026 Apr 7;26:474. doi: 10.1186/s12866-026-05001-y

Technological properties and safety evaluation of bacteriocinogenic Enterococcus strains isolated from raw meat and traditional meat products in Türkiye

Burak Geniş ¹, Yasin Tuncer ^1,^✉

PMCID: PMC13192141 PMID: 41947047

Abstract

Objective

This study aimed to isolate bacteriocin-producing lactic acid bacteria (LAB) from raw meat and traditional Turkish meat products and to evaluate their technological properties and safety for potential use as starter cultures in the meat industry.

Methods

Presumptive LAB were screened for bacteriocin production using indicator strains, and the proteinaceous nature of the antibacterial compounds was confirmed by proteolytic enzymes. The isolates were identified by 16 S rDNA- and species-specific PCR, and their genetic diversity was assessed by RAPD-PCR. Furthermore, bacteriocin stability, bacteriocin type, technological properties, and safety aspects—including antibiotic susceptibility, resistance and virulence genes, hemolytic and gelatinase activities, and biogenic amine production—were evaluated using phenotypic assays and PCR-based methods.

Results

A total of eight presumptive bacteriocin-producing LAB strains were isolated, seven from raw meat and one from sucuk, a traditional Turkish fermented sausage. Proteolytic enzyme treatments confirmed the antibacterial substances produced by these strains were bacteriocins. 16 S rDNA sequencing and PCR-based analyses identified five isolates as Enterococcus faecium and three as Enterococcus mundtii. All strains were found genetically distinct by RAPD-PCR. Bacteriocins produced by all strains exhibited broad pH stability and heat resistance. PCR and Tricine-SDS-PAGE analyses indicated that E. faecium strains produce enterocin B, whereas E. mundtii strains produce mundticin KS. The E. faecium strains showed rapid acid production, while the E. mundtii strains exhibited moderate acid production. None of the strains displayed extracellular proteolytic or lipolytic activities; however, all exhibited esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase activities. Additionally, E. faecium strains demonstrated valine arylamidase and cystine arylamidase activities, which were absent in E. mundtii strains. Nitrate reductase activity was observed in E. mundtii but absent in E. faecium. The strains were generally susceptible to clinically important antibiotics. The multiple antibiotic resistance (MAR) index of E. mundtii strains was lower than that of E. faecium, whose MAR index ranged from 0.22 to 0.26, indicating that these strains were multidrug- resistant. Notably, E. faecium B7.2 and B7.3 lacked detectable antibiotic resistance genes. However, tetL, aph(3ʹ)-IIIa, and ant(4ʹ)-Ia genes were detected in 50% of the strains. All strains did not exhibited hemolytic and gelatinase activities. Virulence factors were not detected in E. mundtii B33.1; however, the remaining seven strains carried one or both of the efaA_fm and acm genes. Furthermore, E. faecium strains decarboxylated tyrosine, whereas E. mundtii exhibited weak activity. The tdc gene was detected in all strains.

Conclusion

These findings indicate that E. mundtii B33.1 may be safely used as a starter or adjunct culture in food processing. In contrast, the remaining seven strains may pose potential health risks if applied as live cultures in food production. However, purified bacteriocins derived from these strains could have promising applications in food preservation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-05001-y.

Keywords: Enterococcus, Enterocin, Raw meat, Dry fermented sausage (Sucuk), Dry cured meat (Pastırma), Technological properties, Safety evaluation

Background

Meat and meat products have long been integral to the human diet due to their rich nutrient profile, which supports human health and growth [1]. However, meat is highly perishable due to intrinsic factors such as pH (5–6.5), high water activity (0.95–0.99), and its nutrient content [2]. Additionally, extrinsic factors, including storage temperature, packaging conditions, endogenous enzymes, humidity, light exposure, and microbial activity, significantly influence the shelf life and quality of meat [3]. Raw meat spoils and becomes unfit for consumption when its natural metabolism or microbial activity alters its physical and chemical properties beyond acceptable levels [4]. In addition, microbial contamination of raw meat and meat products by pathogenic microorganisms presents a significant risk to consumer health [5]. Over the centuries, humans have developed various methods to preserve meat. Among these, fermentation stands out as one of the oldest and most significant techniques. Lactic acid bacteria (LAB) are among the most important microorganisms involved in meat fermentation. While LAB encompass more than 60 genera, Lactobacillus, Leuconostoc, Lactococcus, Weisella, Pediococcus, and Enterococcus are commonly identified in fermented meat products [6]. LAB impart unique sensory characteristics to the final product—including flavor, aroma, and texture—through the breakdown of carbohydrates, fats, and proteins. Additionally, they extend the shelf life of meat by producing antimicrobial peptides, such as bacteriocins, and other antimicrobial metabolites, including hydrogen peroxide, fatty acids, acetoin, diacetyl [7, 8]. LAB specifically reduce the pH of fermented meat products by producing organic acids through carbohydrate fermentation [6], which aids in the coagulation of meat proteins and promotes coloring reactions [8]. In addition to organic acids, LAB produce antibacterial substances such as hydrogen peroxide, diacetyl, biosurfactants, and bacteriocins during growth and fermentation processes, which contribute to the microbiological safety of the end products [9, 10]. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and secreted into the extracellular environment. Bacteriocins produced by LAB primarily target Gram-positive bacteria, including foodborne pathogens [11]. These peptides have effectively been utilized to inhibit pathogens like Salmonella spp., Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus in a range of meat products, enhancing food safety and prolonging the shelf life of the final product [12].

Enterococcus, a genus of LAB, are well-adapted to diverse food systems, particularly traditional cheeses and sausages, owing to their high tolerance to salts and acids. It is believed that these bacteria contribute to the organoleptic properties of these products through lipolysis, proteolysis, and glycolysis [13, 14]. Moreover, some enterococcal strains possess the potential to be used as food preservatives due to their ability to produce bacteriocins, often referred to as enterocins, which are effective against Gram-positive pathogens, particularly L. monocytogenes [15]. On the other hand, enterococci lack Generally Recognized as Safe (GRAS) status from U.S. Food and Drug Administration and Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA) [14]. Certain E. faecalis and E. faecium strains are known as nosocomial opportunistic pathogens, associated with bacteremia, endocarditis, sepsis, and urinary tract infections [16]. The virulence factors responsible for the pathogenicity of enterococci can be categorized into three main types: (i) surface-associated factors, such as aggregation substance, collagen-binding protein, cell wall adhesin, and enterococcal surface protein, that facilitate host cell colonization; (ii) secreted enzymes and toxins, including gelatinase, cytolysin, and hyaluronidase, which contribute to tissue damage; and (iii) sex pheromones, which enhance the conjugative transfer of pheromone-responsive plasmids between cells [17]. Antibiotic resistance is another factor contributing to the pathogenicity of enterococci. These bacteria may exhibit both intrinsic and acquired resistance to several clinically significant classes of antibiotics, including aminoglycosides, fluoroquinolones, glycopeptides, β-lactams, macrolides, streptogramins, and tetracyclines [18]. In addition to virulence factors and antibiotic resistance, enterococcal strains intended for use as probiotics or starter cultures in food should be evaluated for their production of biogenic amines, which may cause food intoxication [19]. Biogenic amines are organic basic compounds that can occur in various foods, including meat products, fish, wine, and cheese. These compounds are typically formed through the microbial decarboxylation of free amino acids and have been linked to adverse health effects, such as raised blood pressure, vomiting, severe headaches, and allergic reactions [20, 21]. Enterococcus species have been previously documented to produce biogenic amines, particularly tyramine; however, these compounds have not yet been implicated in enterococcal disease [20, 22].

Growing consumer preference for high-quality natural foods has intensified research into the use of antibacterial-producing bacteria as starter cultures and the direct incorporation of antibacterial substances derived from these bacteria into food production processes [4, 23]. In recent decades, to mitigate health risks and ensure the production of high-quality traditional products, researchers have increasingly employed autochthonous LAB, known for their antimicrobial properties, in the production of fermented meat products [5]. Various studies conducted in Türkiye have examined Enterococcus strains isolated from milk and dairy products, particularly with respect to their antimicrobial activity and bacteriocin (enterocin) production, and have evaluated selected safety traits, including antibiotic susceptibility, virulence-associated characteristics, and biogenic amine production [24, 25]. In addition, Altınkaynak and Tuncer [26] aimed to characterize a bacteriocin produced by E. mundtii YB6.30 with antilisterial activity isolated from fermented sucuk. Similarly, E. faecium strains isolated from pastırma were reported to produce antimicrobial substances and to exhibit probiotic potential [27]. Studies focusing on enterococci from fermented meat products such as sucuk and pastırma in Türkiye have mainly addressed species distribution and antibiotic resistance profiles [28–30]. However, these studies generally focused on specific attributes—such as antimicrobial activity or antibiotic resistance—without a comprehensive evaluation combining bacteriocin characterization, technological properties, and a full safety assessment, including biogenic amine production. Therefore, the present study extends previous research by integrating bacteriocin characterization with detailed safety and technological evaluations of bacteriocin-producing LAB isolated from both raw meats and traditional Turkish meat products. In this regard, the present study aimed to isolate bacteriocin-producing LAB species from raw meats (lamb/mutton, kid/goat, and veal) as well as from traditional Turkish meat products (sucuk and pastırma), to characterize their bacteriocins, and to evaluate the technological properties and safety of these strains in order to assess their potential as starter cultures in the meat industry.

Materials and methods

Meat and meat product samples

Sixty raw meat samples, consisting of 20 each of lamb/mutton, kid/goat, and calf, as well as 60 traditional Turkish meat product samples (40 sucuk and 20 pastırma), were collected from local butchers in the provinces of Afyonkarahisar, Antalya, Isparta, Kayseri, and Muğla, Türkiye, between October 2019 and February 2020. The provincial distribution of raw meat and meat product samples is shown in Supplementary Material Table S1. Sampling was performed using a random retail sampling design, and butchers were selected based on their willingness to participate and their regular sale of the targeted products. Raw meat samples were obtained as retail meat cuts offered for sale to consumers and consisted of edible muscle tissue. Each raw meat and pastırma sample weighed at least 100 g, while sucuk samples were purchased as whole rings. All samples were individually packed in sterile bags at the point of purchase. The samples were transported to the laboratory in cooler boxes and stored at 4 °C until analysis. Microbiological analyses were initiated within 24 h of collection.

Screening of antimicrobial activity of presumptive LAB isolates

To screening for antimicrobial activity of presumptive LAB isolates, 25 grams of each sample were homogenized with 225 mL of sterile physiological saline solution (0.85% NaCl, w/v) in a Waring blender (Model 8011 ES, Torrington, CT, USA) for 90 seconds, followed by serial dilution. A 100 µL aliquot of each dilution was spread onto de Man, Rogosa, and Sharpe agar (MRS; Biokar Diagnostics, Allonne, France) and 0.5% glucose-containing M17 agar (GM17; Biolife Italiana, Milano, Italy). The MRS and M17 plates were incubated at 37°C and 30°C for 24–48 hours, respectively. Subsequently, the colony surfaces were covered with 5 mL of soft agar (0.5%, w/v) containing 100 µL of L. monocytogenes ATCC 7644 or S. aureus ATCC 25923 (7–8 log CFU/mL) as the indicator bacterium. The presence of zones surrounding the colonies was assessed after 24 hours of incubation at 37°C. Colonies producing inhibition zones larger than 2 mm were aseptically picked and transferred into the corresponding liquid medium. To eliminate possible contamination by the indicator strain and to ensure purity, isolates were streaked onto the solid form of the same medium to obtain single colonies. A single colony was subsequently transferred to liquid medium and subjected to purity control. Finally, the antibacterial activity of the purified isolates was confirmed against the same indicator bacterium according to Geis et al. [31].”

Microscopic morphology of the isolates was detected by Gram staining. Catalase activity of isolates was assessed by using 3% (v/v) hydrogen peroxide (Tekkim Kimya Ltd. Şti., Bursa, Türkiye). S. aureus ATCC 25,923 and E. faecalis ATCC 29,212 were used as positive and negative controls for the catalase test, respectively.

Antibacterial activity spectrum of presumptive bacteriocin-producing LAB and detection of the antibacterial substance’s susceptibility to proteolytic enzymes

The antibacterial activity spectrum of presumptive bacteriocin-producing LAB isolates was evaluated against 27 Gram-positive and Gram-negative indicator bacteria using the sterile toothpick method [32] and the agar well diffusion method [33]. All assays were performed in triplicate. LAB isolates were initially grown in MRS or GM17 broth at 37 and 30 °C for 24 h, respectively. Indicator strains were cultured in their appropriate media at the incubation temperatures specified in Tables 1 and 2. For the sterile toothpick method, overnight LAB cultures were streaked onto MRS or GM17 agar plates and incubated for 24 h at their respective growth temperature (37–30 °C) to allow colony development. Individual colonies were then transferred onto fresh agar plates using sterile toothpicks. After incubation for an additional 24 h at the same temperature, the growing LAB colonies were overlaid with soft agar (0.5%, w/v) previously inoculated with 100 µL of the indicator bacterium (7–8 log CFU/mL). Plates were incubated under appropriate conditions for 24 h, and the presence of clear inhibition zones around the LAB growth area was recorded as indicative of antibacterial activity. For the agar well diffusion assay, overnight LAB cultures were centrifuged at 15,493 × g for 15 min (Sigma 2-16KL, rotor no. 12148, Germany). The culture supernatants were transferred into sterile tubes, and their pH was adjusted to 7.0 using 6 N NaOH. The supernatants were then sterilized by passing them through a 0.45 μm membrane filter (Aisimo Corp., Shanghai, China). 100 µL of the indicator bacterium (7–8 log CFU/mL) was inoculated into 5 mL of soft agar (0.5%, w/v) and overlaid onto agar plates. Wells of 6 mm diameter were aseptically punched into the agar and filled with 100 µL of the sterile, neutralized, cell-free supernatant obtained from the LAB cultures. Plates were incubated at the appropriate temperature for 24 h, and antibacterial activity was determined by measuring the diameter of the inhibition zones surrounding the wells.

Table 1.

Culture media, growth temperature, and sources of indicator strains, and inhibitory activity of bacteriocin-producing strains by using sterile toothpick method

Indicator bacteria	Culture media^a and growth temperature	Source^b	Inhibitory activity of bacteriocin producer strains^{c, d}
Indicator bacteria	Culture media^a and growth temperature	Source^b	B7.2	B7.3	B8.1	B8.2	B9.1	B20.2	B31.2	B33.1
Lactococcus lactis LMG 2907	GM17, 30˚C	NLH	0 ± 0.00^R	0 ± 0.00^N	0 ± 0.00^P	0 ± 0.00^N	0 ± 0.00^N	0 ± 0.00^K	0 ± 0.00^N	0 ± 0.00^M
Lactococcus lactis LMG 2908	GM17, 30˚C	NLH	0 ± 0.00^R	0 ± 0.00^N	0 ± 0.00^P	0 ± 0.00^N	0 ± 0.00^N	0 ± 0.00^K	0 ± 0.00^N	0 ± 0.00^M
Lactococcus lactis LMG 2910	GM17, 30˚C	NLH	11.03 ± 0.06^G	9.00 ± 0.10^E	10.03 ± 0.06^H	9.00 ± 0.00^F	8.00 ± 0.00^G	12.47 ± 0.06^H	15.00 ± 0.00^I	16.47 ± 0.06^I
Lactococcus lactis LMG 1363	GM17, 30˚C	NLH	11.00 ± 0.00^G	11.00 ± 0.10^C	12.03 ± 0.06^D	12.00 ± 0.00^C	11.00 ± 0.00^C	0 ± 0.00^K	0 ± 0.00^N	0 ± 0.00^M
Listeria innocua LMG 2813	GM17, 30˚C	NLH	7.03 ± 0.06^{e, K}	8.47 ± 0.06^{e, F}	8.00 ± 0.00^{e, J}	8.00 ± 0.00^{e, G}	8.00 ± 0.00^{e, G}	13.00 ± 0.00^H	16.03 ± 0.06^H	17.00 ± 0.10^H
Enterococcus faecalis LMG 2602	GM17, 30˚C	NLH	11.00 ± 0.00^G	11.03 ± 0.06^C	12.00 ± 0.00^E	13.00 ± 0.10^B	11.00 ± 0.00^C	3.00 ± 0.00^J	0 ± 0.00^N	0 ± 0.00^M
Enterococcus faecalis ATCC 29,212	MRS, 37˚C	SDÜBGL	5.50 ± 0.10^P	7.00 ± 0.00^I	8.00 ± 0.00^J	7.03 ± 0.06^H	7.47 ± 0.06^H	17.03 ± 0.06^E	18.00 ± 0.10^G	19.00 ± 0.00^F
Enterococcus faecalis ATCC 51,299	MRS, 37˚C	SDÜBGL	15.03 ± 0.06^C	13.03 ± 0.06^A	15.03 ± 0.06^B	12.00 ± 0.00^C	14.00 ± 0.10^B	16.00 ± 0.00^F	19.00 ± 0.00^F	18.03 ± 0.06^G
Enterococcus faecium ATCC 51,559	MRS, 37˚C	SDÜBGL	15.47 ± 0.06^{e, B}	12.00 ± 0.00^{e, B}	10.50 ± 0.10^{e, G}	0 ± 0.00^N	8.00 ± 0.00^{e, G}	18.07 ± 0.12^D	20.47 ± 0.06^C	20.00 ± 0.00^E
Enterococcus faecalis LMG 2708	GM17, 30˚C	NLH	10.00 ± 0.00^H	8.03 ± 0.06^G	8.00 ± 0.00^J	6.47 ± 0.06^I	8.00 ± 0.00^G	15.47 ± 0.06^F	16.00 ± 0.00^H	17.47 ± 0.06^H
Lactobacillus plantarum LMG 2003	MRS, 37˚C	NLH	17.00 ± 0.10^{e, A}	13.00 ± 0.00^A	16.00 ± 0.10^{e, A}	15.00 ± 0.00^{e, A}	15.00 ± 0.00^{e, A}	0 ± 0.00^K	0 ± 0.00^N	0 ± 0.00^M
Pediococcus pentacaceus LMG 2001	TSBYE, 37˚C	NLH	13.97 ± 0.06^E	11.00 ± 0.00^{e, C}	12.03 ± 0.06^D	5.00 ± 0.00^K	10.03 ± 0.06^{e, D}	4.00 ± 0.00^J	10.00 ± 0.00^{e, J}	10.00 ± 0.00^{e, J}
Listeria monocytogenes ATCC 19,115	TSBYE, 37˚C	SDÜBGL	5.00 ± 0.00^R	6.00 ± 0.00^J	5.00 ± 0.00^N	6.00 ± 0.00^J	5.03 ± 0.06^L	4.47 ± 0.06^I	4.00 ± 0.00^M	4.00 ± 0.00^L
Listeria monocytogenes ATCC 7644	TSBYE, 37˚C	SDÜBGL	14.47 ± 0.06^D	13.03 ± 0.06^A	13.47 ± 0.06^C	13.00 ± 0.00^B	8.00 ± 0.00^{e, G}	20.00 ± 0.10^B	19.03 ± 0.06^F	21.00 ± 0.00^C
Escherichia coli LMG 3083 (ETEC)	TSBYE, 37˚C	NLH	9.00 ± 0.00^{e, I}	10.03 ± 0.06^{e, D}	9.00 ± 0.00^{e, I}	10.00 ± 0.10^{e, E}	8.03 ± 0.06^{e, F}	18.50 ± 0.10^{e, C}	18.03 ± 0.06^{e, G}	21.03 ± 0.06^{e, C}
Escherichia coli ATCC 25,922	TSBYE, 37˚C	SDÜBGL	5.47 ± 0.06^{e, P}	4.47 ± 0.06^{e, M}	7.00 ± 0.00^{e, K}	6.97 ± 0.06^{e, H}	5.00 ± 0.00^{e, L}	20.00 ± 0.10^{e, B}	20.47 ± 0.15^{e, C}	22.03 ± 0.06^{e, A}
Salmonella Typhimurium ATCC 14,028	TSBYE, 37˚C	SDÜBGL	8.00 ± 0.00^J	5.00 ± 0.00^L	6.00 ± 0.00^M	4.53 ± 0.06^L	8.00 ± 0.00^G	20.03 ± 0.06^B	15.97 ± 0.06^H	18.50 ± 0.10^G
Salmonella Enteritidis ATCC 13,076	TSBYE, 37˚C	SDÜBGL	8.00 ± 0.00^J	7.00 ± 0.00^I	7.00 ± 0.00 ^K	7.00 ± 0.00 ^H	8.00 ± 0.00^G	4.53 ± 0.06^I	5.00 ± 0.00^K	5.00 ± 0.00^K
Staphylococcus aureus ATCC 25,923	TSBYE, 37˚C	SDÜBGL	12.00 ± 0.00^{e, F}	9.97 ± 0.06^{e, D}	11.03 ± 0.06^{e, F}	11.00 ± 0.00^{e, D}	9.03 ± 0.06^{e, E}	14.00 ± 0.00^H	20.03 ± 0.06 ^E	22.00 ± 0.00^B
Staphylococcus aureus ATCC 43,300	TSBYE, 37˚C	SDÜBGL	7.00 ± 0.00^L	7.00 ± 0.00^I	9.00 ± 0.00^I	7.03 ± 0.06 ^H	5.97 ± 0.06^K	21.0 ± 0.00^{e, A}	20.97 ± 0.06^{e, B}	21.97 ± 0.06^{e, B}
Staphylococcus aureus ATCC 6538	TSBYE, 37˚C	SDÜBGL	0 ± 0.00^R	8.50 ± 0.00 ^F	9.00 ± 0.00^I	7.00 ± 0.00 ^H	6.00 ± 0.00^K	17.03 ± 0.06^{e, E}	19.00 ± 0.00^{e, F}	20.03 ± 0.06^{e, E}
Staphylococcus carnosus LMG 2709	TSBYE. 37˚C	NLH	6.03 ± 0.06^N	5.00 ± 0.00^L	6.50 ± 0.00^L	4.00 ± 0.00^M	0 ± 0.00^N	15.03 ± 0.06^{e, G}	18.00 ± 0.00^{e, G}	19.00 ± 0.10^{e, F}
Bacillus cereus ATCC 10,876	TSBYE. 37˚C	SDÜBGL	6.47 ± 0.06^M	7.0 ± 0.00^I	7.00 ± 0.00 ^K	7.00 ± 0.00 ^H	6.50 ± 0.00^J	0 ± 0.00^K	4.50 ± 0.10^L	5.47 ± 0.06^J
Bacillus cereus ATCC 11,778	TSBYE. 37˚C	SDUBB	10.00 ± 0.00^{e, H}	8.00 ± 0.00^G	8.00 ± 0.00^J	6.00 ± 0.00^J	5.97 ± 0.15^{e, K}	15.03 ± 0.06^{e, G}	22.00 ± 0.10^{e, A}	21.00 ± 0.00^{e, C}
Bacillus subtilis ATCC 6051	TSBYE. 37˚C	SDÜBB	7.00 ± 0.00^L	7.03 ± 0.06^H	6.00 ± 0.00^M	6.47 ± 0.15^I	7.03 ± 0.06^I	5.00 ± 0.00^H	4.97 ± 0.06^K	4.97 ± 0.06^K
Pseudomonas aeruginosa ATCC 15,442	TSBYE. 37˚C	SDÜBGL	7.00 ± 0.00^L	7.00 ± 0.00^I	7.00 ± 0.00 ^K	5.00 ± 0.00^K	7.00 ± 0.00^I	17.97 ± 0.06^{e, D}	19.00 ± 0.00^{e, F}	21.00 ± 0.00^{e, C}
Pseudomonas aeruginosa ATCC 27,853	TSBYE. 37˚C	SDÜBB	6.00 ± 0.00^{e, O}	5.50 ± 0.00^K	4.47 ± 0.06 ^O	4.00 ± 0.00^M	4.00 ± 0.00^M	17.03 ± 0.06^{e, E}	20.97 ± 0.06^{e, B}	22.00 ± 0.00^{e, B}

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^aTSBYE: Tryptic soy broth (containing 0.5% yeast extract); MRS: de Man Rogosa and Sharpe broth; GM17: M17 broth (containing 0.5% glucose)

^bRSK: Refik Saydam Hygiene Institute Culture Collection Ankara/Türkiye; NLH: Laboratory of Microbial Gene Technology, Department of Genetics and Biochemistry, Agricultural University of Norway (NLH). Ås/Norway); SDÜBGL: Laboratory of Bacterial Genetics, Department of Food Engineering, Süleyman Demirel University, Isparta/Türkiye; SDÜBB: Department of Biology, Süleyman Demirel University, Isparta/Türkiye

^cValues represent inhibition zone diameters expressed as mean ± standard deviation (mm)

^dThe capital letters to the right of the mean indicate the difference between the antibacterial activities of each Enterococcus strain against indicator bacteria

^eFaint zone of inhibition

Table 2.

Culture media, growth temperature, and sources of indicator strains, and inhibitory activity of bacteriocin-producing strains by using the agar well diffusion method

Indicator bacteria	Culture media^a and growth temperature	Source^b	Inhibitory activity of bacteriocin producer strains^{c, d}
Indicator bacteria	Culture media^a and growth temperature	Source^b	B7.2	B7.3	B8.1	B8.2	B9.1	B20.2	B31.2	B33.1
Lactococcus lactis LMG 2907	GM17, 30˚C	NLH	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Lactococcus lactis LMG 2908	GM17, 30˚C	NLH	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Lactococcus lactis LMG 2910	GM17, 30˚C	NLH	9.50 ± 0.10^E	10.57 ± 0.12^F	10.00 ± 0.00^H	10.00 ± 0.10^H	10.47 ± 0.06^G	8.47 ± 0.06^G	10.53 ± 0.06^G	10.00 ± 0.06^G
Lactococcus lactis LMG 1363	GM17, 30˚C	NLH	7.00 ± 0.00^I	9.03 ± 0.06^I	10.50 ± 0.10^G	11.00 ± 0.00^G	11.00 ± 0.00^F	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Listeria innocua LMG 2813	GM17, 30˚C	NLH	11.47 ± 0.06^C	12.03 ± 0.06^D	11.03 ± 0.06^F	11.47 ± 0.06^F	12.00 ± 0.00^D	10.47 ± 0.06^F	11.00 ± 0.00^F	11.03 ± 0.06^F
Enterococcus faecalis LMG 2602	GM17, 30˚C	NLH	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Enterococcus faecalis ATCC 29,212	MRS, 37˚C	SDÜBGL	9.00 ± 0.00^F	10.00 ± 0.00^H	9.03 ± 0.06^I	9.97 ± 0.06^H	10.47 ± 0.06^G	11.00 ± 0.00^E	12.00 ± 0.00^D	12.00 ± 0.00^D
Enterococcus faecalis ATCC 51,299	MRS, 37˚C	SDÜBGL	11.50 ± 0.50^C	14.00 ± 0.00^C	13.50 ± 0.50^C	13.00 ± 0.00^C	13.03 ± 0.06^C	12.00 ± 0.00^C	13.50 ± 0.00^B	14.00 ± 0.00^B
Enterococcus faecium ATCC 51,559	MRS, 37˚C	SDÜBGL	8.00 ± 0.00^H	10.47 ± 0.06^G	11.00 ± 0.00^F	11.47 ± 0.06^F	11.50 ± 0.00 ^E	12.47 ± 0.06^B	13.00 ± 0.00^C	13.43 ± 0.12^C
Enterococcus faecalis LMG 2708	GM17, 30˚C	NLH	9.93 ± 0.12^D	12.03 ± 0.06^D	10.03 ± 0.15^H	12.10 ± 0.10^{e, D}	12.00 ± 0.00^{e, D}	11.00 ± 0.00^E	11.50 ± 0.00^E	12.00 ± 0.00^D
Lactobacillus plantarum LMG 2003	MRS, 37˚C	NLH	8.53 ± 0.06^{e, G}	12.00 ± 0.00 ^{e, D}	11.00 ± 0.10^{e, F}	12.00 ± 0.00^{e, E}	12.00 ± 0.00^{e, D}	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Pediococcus pentacaceus LMG 2001	TSBYE, 37˚C	NLH	9.00 ± 0.00^F	11.00 ± 0.00^E	11.50 ± 0.00^E	11.03 ± 0.06^G	12.00 ± 0.00^{e, D}	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Listeria monocytogenes ATCC 19,115	TSBYE, 37˚C	SDÜBGL	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Listeria monocytogenes ATCC 7644	TSBYE, 37˚C	SDÜBGL	16.00 ± 0.10^B	17.50 ± 0.00^B	17.00 ± 0.00^B	17. 03 ± 0.00^B	17.03 ± 0.15^B	18.00 ± 0.00^A	19.03 ± 0.06^A	19.47 ± 0.06^A
Escherichia coli LMG 3083 (ETEC)	TSBYE, 37˚C	NLH	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Escherichia coli ATCC 25,922	TSBYE, 37˚C	SDÜBGL	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Salmonella Typhimurium ATCC 14,028	TSBYE, 37˚C	SDÜBGL	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Salmonella Enteritidis ATCC 13,076	TSBYE, 37˚C	SDÜBGL	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Staphylococcus aureus ATCC 25,923	TSBYE, 37˚C	SDÜBGL	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Staphylococcus aureus ATCC 43,300	TSBYE, 37˚C	SDÜBGL	24.03 ± 0.06^{e, A}	24.00 ± 0.00^{e, A}	23.00 ± 0.00^{e, A}	24.03 ± 0.06^{e, A}	26.00 ± 0.10^{e, A}	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Staphylococcus aureus ATCC 6538	TSBYE, 37˚C	SDÜBGL	0 ± 0.00^J	11.00 ± 0.00^{e, E}	12.00 ± 0.00^{e, D}	7. 00 ± 0.00^{e, J}	13. 03 ± 0.06^{e, C}	8.53 ± 0.06^{e, G}	11.50 ± 0.10^E	10.00 ± 0.00^F
Staphylococcus carnosus LMG 2709	TSBYE. 37˚C	NLH	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Bacillus cereus ATCC 10,876	TSBYE. 37˚C	SDÜBGL	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Bacillus cereus ATCC 11,778	TSBYE. 37˚C	SDUBB	0 ± 0.00^J	0 ± 0.00^J	9.00 ± 0.00^{e, I}	8. 53 ± 0.06^{e, I}	11. 00 ± 0.00^{e, F}	11.50 ± 0.00^{e, D}	12. 00 ± 0.00^{e, D}	11. 00 ± 0.00^{e, E}
Bacillus subtilis ATCC 6051	TSBYE. 37˚C	SDÜBB	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Pseudomonas aeruginosa ATCC 15,442	TSBYE. 37˚C	SDÜBGL	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G
Pseudomonas aeruginosa ATCC 27,853	TSBYE. 37˚C	SDÜBB	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^J	0 ± 0.00^K	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^H	0 ± 0.00^G

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^aTSBYE: Tryptic soy broth (containing 0.5% yeast extract); MRS: de Man Rogosa and Sharpe broth; GM17: M17 broth (containing 0.5% glucose)

^cValues represent inhibition zone diameters expressed as mean ± standard deviation (mm)

^dThe capital letters to the right of the mean indicate the difference between the antibacterial activities of each Enterococcus strain against indicator bacteria

^eFaint zone of inhibition

The susceptibility of the antibacterial substance produced by the isolates to proteolytic enzymes—pepsin, proteinase K, trypsin, and α-chymotrypsin—was assessed using the method described by Ryan et al. [34]. Catalase was employed to eliminate the antibacterial activity caused by hydrogen peroxide. The enzymes were procured from Sigma-Aldrich (USA). Application of the proteolytic enzymes resulted in a crescent-shaped zone of lost antibacterial activity, indicating that the synthesized antibacterial substance was a bacteriocin.

Identification of bacteriocin-producing isolates

Genomic DNA was isolated from 0.5 mL of an overnight culture (18–24 h old) of isolates using the method recommended by Cancilla et al. [35]. Cells were pelleted by centrifugation at 15,493 × g for 5 min at 4 °C using a refrigerated centrifuge (Sigma 2-16KL, rotor no. 12148). The pellets were resuspended in 0.5 mL of lysis buffer and incubated in a water bath (Nüve NB9, Türkiye) at 37 °C for one hour. Subsequently, 30 µL of sodium dodecyl sulfate (SDS, 10% w/v) was added, and the tubes were incubated in a water bath (Nüve NB5, Türkiye) at 80 °C for 5 min. Following this, 0.7 mL of a phenol-chloroform (1:10, v/v) solution was added, and the tubes were centrifuged under the same parameters as given above. The upper phase was transferred to new tubes, and 0.7 mL of cold 2-propanol was added. The tubes were then centrifuged again under the same conditions. The genomic DNA pellets were dissolved in 50 µL of Tris-EDTA (pH 8.0) and stored at − 20 °C until further use.

The bacteriocin-producing isolates were identified using molecular methods based on PCR targeting specific regions of the 16 S rRNA gene [36], the Enterococcus genus-specific tuf gene [37], and the Enterococcus species-specific sodA gene [38]. Details of the primers and PCR protocols used for identification are provided in Supplementary Material Table S2. For gene amplification, a 50 µL PCR mix was prepared, consisting of 25 µL of 2× PCR master mix (ABM, Canada), 20 µL of nuclease-free water, 1 µL of each primer (100 pM/µL), and 3 µL of genomic DNA. Amplification was carried out using a gradient thermal cycler (TurboCycler 2, Blue-Ray Biotech Corp., Taipei City, Taiwan). PCR products were electrophoresed on 1 or 2% (w/v) agarose gels in Tris-Acetate-EDTA (TAE) buffer (Wisent Inc., Canada). The gels were stained in a 0.2 µg/mL ethidium bromide solution for 30–45 min and then photographed under a UV transilluminator (ECX-F20.M, Vilber Lourmat, France) using a digital camera (Nikon D5100, Japan). Fragment sizes were determined using the Genesta™ 100-bp DNA marker (GeneAll Bldg., GA-010, Seoul, Korea).

Genotyping of Enterococcus isolates by RAPD-PCR

Bacteriocin-producing Enterococcus isolates were genotyped using the RAPD-PCR technique with the M13 primer (5’-GAG GGT GGC GGT TCT-3’) as suggested by Rossetti and Giraffa [39]. RAPD-PCR was performed in 50 µL reaction mixtures prepared by combining 25 µL of Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific, F-548 L), 17 µL of nuclease-free water, 5 µL of primer, and 3 µL of template DNA. A total of 40 amplification cycles were conducted, consisting of denaturation at 94 °C for 1 min, annealing at 42 °C for 20 s, and elongation at 72 °C for 2 min. The RAPD-PCR products were electrophoresed on a 2% (w/v) agarose gel in TAE buffer (Wisent). The gel was stained and photographed as described previously. The GelJ program [40] was used to analyze genetic similarities between the isolates. A dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Means (UPGMA) based on the Dice similarity index.

The effect of different enzyme, heat and pH treatments on bacteriocin activity

The cell-free supernatant (CFS) was obtained by centrifuging overnight cultures of the isolates at 3059 x g for 15 min (Sigma 2-16P, rotor no. 12141). These CFSs were used to evaluate the effects of various enzyme, heat, and pH treatments on bacteriocin activity. The CFSs were neutralized with 6 N NaOH and sterilized by passing them through 0.45 μm membrane filters (Aisimo, China). For enzyme treatment, proteinase K, trypsin, α-chymotrypsin, pepsin, α-amylase, lipase, catalase, or lysozyme were added to the CFSs at a final concentration of 1 mg/mL and incubated at 37 °C for two hours. Enzyme activities were then stopped by heat treatment at 100 °C for five minutes. Enzyme-untreated CFSs were used as controls. To determine the effect of heat treatment on bacteriocin activity, neutralized CFSs were heated at 100 °C for 5, 10, 15, and 20 min and autoclaved at 121 °C for 15 min. Unheated CFSs were used as controls. To determine the effect of pH on bacteriocin activity, the pH of the CFSs was adjusted between 2.0 and 11.0 using 6 N NaOH or 6 N HCl before filter sterilization, and then they were kept at 4 °C for 24 h. Untreated CFSs were used as controls. The remaining activity of the CFSs was measured using the critical dilution method according to Franz et al. [41].

Detection of enterocin-encoding genes by PCR

PCR was used to investigate the structural genes of well-known enterocins, including enterocin A, enterocin B, enterocin P, enterocin Q, enterocin L50A/B, enterocin 1071 A/B, bacteriocin 31, enterocin AS48, enterocin CRL35, and mundticin KS [42–45]. PCR reaction mixtures were prepared as described above. The PCR primers, PCR protocols, and product sizes used for screening the enterocin genes are listed in Table S2. The PCR fragments were subjected to electrophoresis on 2% (w/v) agarose gels in TAE buffer. The GenestaTM 100-bp DNA ladder (Korea) was used as a molecular marker. E. faecium EYT17, which contains the entA, entB, and entP genes [46] and E. mundtii YB6.30, which harbors the munKS gene [26], were used as positive controls.

Purification of bacteriocins, tricine-SDS-PAGE and detection of active protein bands

The bacteriocins were purified using slight modifications to the protocol described by Foulquié Moreno et al. [47]. To summarize, ammonium sulfate was gradually added to 100 mL of neutralized culture supernatants of enterocin-producing isolates until saturation reached 60%, and then kept in the refrigerator overnight. Subsequently, the samples were centrifuged at 4 °C at 15,294 × g for 30 min (Sigma 2-16KL, rotor no. 12181). The liquid phase was decanted, and the pellets at the bottom and on the walls were dried at ambient temperature. The dehydrated pellets were dissolved in sodium phosphate buffer and extracted with methanol/chloroform (1:2, v/v) at 4 °C for one hour. The resulting mixture was centrifuged at 15,294 × g for 45 min (Sigma 2-16KL, rotor no. 12139). The pellets were dehydrated at ambient temperature and dissolved in 500 µL of sterile ultra-pure water. Partially purified bacteriocins were dialyzed overnight using a 1000 Da dialysis membrane (Spectra/Por^®, USA) and stored at -20 °C.

The tricine-SDS-PAGE technique was used to determine the molecular sizes of partially purified bacteriocins [48]. Electrophoresis was performed using a vertical gel system (Sigma-Aldrich, USA), consisting of a 10% stacking gel and a 16% resolving gel. An electric current of 30 volts was applied to the stacking gel and 90 volts to the resolving gel for approximately 20–22 h. For molecular size determination, 22 µL of the bacteriocin sample was loaded into the gel wells, while 10 µL of the bacteriocin sample was loaded for bacteriocin activity detection. The Thermo Scientific Spectra Multicolor Low Range Protein Ladder (catalog number 26628) was used as a protein standard. After the electrophoretic separation of the bacteriocin samples was completed, the gel was divided into two parts. The section for molecular size determination was stained with silver nitrate, while the unstained section was used to detect active protein bands, after being washed with sterile distilled water for 24 h. To identify active protein bands, the washed gel surface was covered with 60 mL of MRS soft agar (0.8%, w/v) containing indicator bacteria, either E. faecalis ATCC 51,299 or E. faecium ATCC 51,559. The gel was incubated at 37 °C for 18 h, and the inhibition zone formed on the gel was then checked.

Technological properties of bacteriocin-producing Enterococcus strains

Acid production

The acid production ability of the strains was determined by inoculating 1% (v/v) of 18-hour-old cultures, with a cell density of approximately 7–8 log CFU/mL, into MRS broth. The inoculated samples were incubated at 37 °C, and the culture pH was measured at 0, 6, and 24 h using a pH meter (WTW 3110, Germany). Acid production ability of the cultures was calculated by considering the difference (∆pH) between the initial pH value and the pH value after incubation [49]. The strains were classified as fast (ΔpH > 1.5), moderate (1.0 < ΔpH < 1.5), or slow (ΔpH < 1.0) acid producers according to Bradley et al. [50].

Proteolytic and lipolytic activities

The proteolytic activity of the strains was determined on calcium caseinate agar (Sigma-Aldrich 21065, Switzerland), according to Martín et al. [51]. After incubating the Petri plates at 37 °C for 72 h, the development of a zone around the colony was considered a sign of proteolytic activity.

The lipolytic activity of the strains was assessed on Luria-Bertani (LB, Sigma-Aldrich, 28713) agar supplemented with 0.2% (w/v) CaCl₂ (Isolab, Germany) and 0.1% (w/v) Tween 80 (Sigma-Aldrich, P4780) [52], as well as on spirit blue agar (BD DifcoTM 295020, France) supplemented with lipase reagent (BD DifcoTM 215335) [53]. The LB agar and spirit blue agar plates were incubated at 37 °C for 48 and 72 h, respectively. The formation of an opaque zone around the colony on both media was considered a positive result.

Enzyme profiles

The enzyme profiles of the strains were examined using the API^® ZYM kit (BioMérieux, Marcy-l’Etoile, France). The color variations and intensities in the strips were assessed on a scale from 0 to 5, following the manufacturer’s instructions. Scores of 3, 4, or 5 were classified as positive reactions, while the remaining scores indicated negative reactions.

Nitrate reductase activity

The nitrate reductase activity of the strains was evaluated on YT agar containing 0.1% (w/v) KNO₃, according to Miralles et al. [54]. After coating the colonies with nitrate reagent A (Fluka 38497, Buchs, Switzerland) and nitrate reagent B (Fluka 39441), a positive result was indicated by a red coloration of the colony. Nitrate reductase-positive Escherichia coli ATCC 25,922 and Salmonella Typhimurium ATCC 14,028 strains were used as positive controls.

Safety properties of bacteriocin-producing Enterococcus strains

Antibiotic resistance: phenotypical and genotypical determination

The antibiotic resistance profiles of the strains were determined using the disk diffusion method, as described by Cariolato et al. [55]. Commercial antibiotic disks representing various groups—including aminoglycosides (gentamicin 30 and 120 µg, streptomycin 300 µg), ansamycins (rifampin 5 µg), carbapenems (imipenem 10 µg), fluoroquinolones (ciprofloxacin 5 µg, levofloxacin 5 µg, norfloxacin 10 µg), glycopeptides (vancomycin 30 µg), lipoglycopeptides (teicoplanin 30 µg), macrolides (erythromycin 15 µg), nitrofurantoins (nitrofurantoin 100 and 300 µg), oxazolidinones (linezolid 30 µg), phenicols (chloramphenicol 30 µg), penicillins (ampicillin 2 and 10 µg, penicillin G 10U), streptogramins (quinupristin-dalfopristin 15 µg), and tetracyclines (doxycycline 30 µg, minocycline 30 µg, tigecycline 15 µg, tetracycline 30 µg)—were obtained from Oxoid Ltd. (Basingstoke, England). The results of the disk diffusion test were interpreted as susceptible, intermediate, or resistant, following the guidelines of the European Committee on Antimicrobial Susceptibility Testing [56] and the Clinical and Laboratory Standards Institute [57]. E. faecalis ATCC 29,212 was used as the control strain. The MAR index for each isolate was calculated by dividing the number of antibiotics to which it was resistant by the total number of antibiotics tested. In this study, multidrug resistance (MDR) was defined as the ability to resist at least one antibiotic from three or more distinct antibiotic classes [58].

The presence of resistance genes for erythromycin (ermA, ermB, ermC), tetracycline (tetK, tetL, tetM, tetO, tetS), high-level aminoglycosides (aac(6ʹ)-Ie-aph(2ʹʹ)-Ia, aph(3ʹ)-IIIa, ant(4ʹ)-Ia, ant(6ʹ)-Ia, aph(2ʹʹ)-Ib, aph(2ʹʹ)-Ic, aph(2ʹʹ)-Id), and vancomycin (vanA, vanB, vanC1, vanC2, vanC3, vanD, vanE, vanG) was determined using specific primers [59–64]. The primer pairs and PCR conditions used for the identification of antibiotic resistance genes are provided in Table S2. E. gallinarum DYE22 (vanC1⁺, vanD⁺), E. casseliflavus DYE26 (tetS⁺), E. casseliflavus DYE43 (tetL⁺, tetM⁺, vanC2⁺, vanC3⁺), E. gallinarum DYE45 (ermA⁺, ermB⁺) [65], E. durans RG36.3 (aph(3ʹ)-IIIa⁺, aph(2ʹʹ)-Ic⁺) [66], E. faecium MSM104.1 (ant(4ʹ)-Ia⁺, ant(6ʹ)-Ia⁺, aph(2ʹʹ)-Ib⁺) [67], E. faecium FYE41 (ermC⁺) [28], E. faecalis ATCC 51,299 (aac(6ʹ)-Ie-aph(2ʹʹ)-Ia⁺, vanB⁺), and E. faecium ATCC 51,559 (vanA⁺) strains were used as controls. PCR amplification was carried out in a 50 µL PCR mixture prepared as previously described, using 2× PCR master mix (ABM, Canada). Electrophoresis of PCR fragments was performed on 1.5% (w/v) agarose gels, and gel images were captured under UV light. The O’GeneRuler™ 100 bp DNA marker (Thermo Fisher Scientific #SM1153, USA) was used as a molecular marker to determine the sizes of the PCR fragments.

Virulence factors: phenotypical and genotypical determination

The hemolytic and gelatinase activities of the strains were assessed as part of the phenotypic determination of their virulence factors. Hemolytic activity was evaluated on Columbia agar (Liofilchem, Roseto degli Abruzzi, Italy) supplemented with 5% (v/v) defibrinated sheep blood. The results were classified as β-hemolytic, α-hemolytic, or γ-hemolytic, as described by Cariolato et al. [55]. The β-hemolytic strain S. aureus ATCC 29,213 served as the control.

Gelatinase activity was tested using Todd-Hewitt agar (Acumedia LAB M) containing 3% (w/v) gelatin (Merck), following the method outlined by Eaton and Gasson [68]. E. faecalis RG22.4, a gelatinase-positive strain, was used as the control [66].

The presence of genes encoding various virulence factors, including gelatinase (gelE), cell wall adhesins (efaA_fm, efaA_fs), extracellular surface proteins (esp_fm, esp_fs), sex pheromones (cpd, cob, ccf, cad), collagen-binding protein (ace, acm), aggregation protein (agg), cytolysin (cylM, cylB, cylA), and hyaluronidase (hyl), was investigated in the strains [44, 68–71]. The primer pairs and PCR conditions employed for the identification of virulence factor genes are detailed in Table S2. PCR amplification was conducted in a 50 µL reaction mixture prepared as described above. E. faecalis ATCC 29,212 (aag⁺, ace⁺, cad⁺, ccf⁺, cob⁺, cpd⁺, cylM⁺, cylB⁺, cylA⁺, esp_fm⁺, esp_fs⁺, gelE⁺), E. faecalis ATCC 51,299 (efaA_fs⁺), and E. faecium ATCC 51,559 (acm⁺, efaA_fm⁺, hyl⁺) strains were used as controls. The resulting PCR products were analyzed via electrophoresis on 1.5% (w/v) agarose gels, and the gel images were documented as previously mentioned. The O’GeneRuler™ 100 bp DNA ladder (Thermo Fisher Scientific, #SM1153, USA) served as a molecular marker for size determination of the PCR fragments.

Biogenic amine production: phenotypical and genotypical determination

The production of biogenic amines by the strains was evaluated using amino acid decarboxylase agar supplemented with precursor amino acids—histidine, lysine, ornithine, or tyrosine (Merck)—at a final concentration of 1% (w/v), following the protocol recommended by Bover-Cid and Holzapfel [72]. The strains were initially subcultured five times in MRS broth containing 0.1% (w/v) of the precursor amino acid. Subsequently, 10 µL of the final cultures were inoculated onto the amino acid decarboxylase medium. After incubation at 37 °C for 2–5 days, biogenic amine production was assessed. Biogenic amine formation was indicated by a color change from yellow to purple around colonies on media containing lysine, ornithine, or histidine, or by the appearance of precipitation around colonies on media containing tyrosine. Basal decarboxylase agar without precursor amino acids served as a negative control.

The presence of amino acid decarboxylase genes, including histidine (hdc), lysine (ldc), ornithine (odc), and tyrosine (tdc), was investigated using PCR with specific primers, as recomemded by de Las Rivas et al. [73]. The primer pairs and PCR conditions employed for the identification of amino acid decarboxylase genes are detailed in Table S2. The amplified PCR products were electrophoresed on 1.5% (w/v) agarose gels and visualized as previously described. The tyraminogenic E. faecium NYE54 (tdc⁺) was utilized as a positive control during phenotypic and genotypic assessments [74].

Statistical analysis

Antibacterial activity and acid production assays were performed in triplicate, and the results were expressed as mean ± standard deviation. For antibacterial activity data, normality and homogeneity of variance were assessed using the Anderson–Darling test and Levene’s test, respectively. Since the data did not meet parametric test assumptions, differences among strains in terms of antibacterial spectrum were evaluated using the Kruskal–Wallis nonparametric test. When significant differences were detected, pairwise comparisons were performed using Dunn’s test with Bonferroni correction. The Mann–Whitney U test was used to compare antibacterial activity detection methods. Acid production (ΔpH) was analyzed using two-way repeated-measures analysis of variance (ANOVA) within the General Linear Model (GLM) framework, with time as the within-subject factor. When significant effects were observed, mean comparisons were performed using the least significant difference (LSD) post hoc test. All statistical analyses were performed at a significance level of P < 0.05.

Results

Screening of presumptive bacteriocin-producing isolates

As a result of the presumptive bacteriocin-producing isolate screening test, a total of 26 colonies that exhibited zone formation larger than 2 mm against the indicator bacteria L. monocytogenes ATCC 7644 or S. aureus ATCC 25,923 were isolated as presumptive bacteriocin-producing bacteria. Following the antimicrobial activity control test, eight colonies that showed no antibacterial activity against the indicators were eliminated. The remaining 18 isolates were found to be Gram-positive (17 cocci and one bacilli) and catalase-negative, indicating that they are presumptive LAB isolates. Among the 18 isolates, nine originated from beef, five from sucuk, three from mutton, and one from goat; however, no antimicrobial activity–producing strains were obtained from pastırma samples. The origins and morphological characteristics of the 18 selected LAB isolates are summarized in Supplementary Table S3.

Antibacterial activity spectrum of LAB and detection of the antibacterial substance’s susceptibility to proteolytic enzymes

The sterile toothpick method showed that all 18 selected LAB isolates produce inhibition zones ranging from 3 to 22 mm against the tested indicator bacteria. The isolates exhibited antimicrobial activity against a broad range of pathogens, including L. monocytogenes (Fig. 1A), E. coli, S. Typhimurium (Fig. 1B), S. aureus, Bacillus cereus, and Pseudomonas aeruginosa, as well as LAB. In the agar well diffusion assay, only the culture supernatants of isolates B7.2, B7.3, B8.1, B8.2, B9.1, B20.2, B31.2, and B33.1 produced inhibition zones, with diameters ranging from 7 to 26 mm (data not shown). Based on these findings, ten isolates that showed limited activity in the toothpick method and no detectable activity in the well diffusion assay were excluded from further analyses. The antibacterial spectra of the eight selected LAB isolates (B7.2, B7.3, B8.1, B8.2, B9.1, B20.2, B31.2, and B33.1,) are presented in Table 1 (sterile toothpick method) and Table 2 (agar well diffusion method). Overall, inhibition patterns were strongly strain- and method-dependent, and the differences among strains in terms of antibacterial spectrum were statistically significant (P < 0.05). Strains B20.2, B31.2, and B33.1 generally exhibited broader inhibitory activity and larger inhibition zones against several indicator bacteria, whereas strains B7.2, B7.3, B8.1, B8.2, and B9.1 showed narrower and more variable inhibition profiles. Notably, across both assays, all LAB isolates displayed their highest inhibitory activity against E. faecalis ATCC 51,299, E. faecium ATCC 51,559, and L. monocytogenes ATCC 7644, with significantly larger inhibition zones compared to other indicator strains (P < 0.05).

Fig. 1 — Antibacterial activity test results of some enterococcal isolates against *L. monocytogenes* ATCC 7644 (A) and S. Typhimurium ATCC 14,028 (B) strains

The proteolytic enzyme treatment revealed that the antibacterial substance produced by these eight selected isolates lost its activity when treated with proteinase K, trypsin, α-chymotrypsin, and pepsin but was not affected by catalase (Fig. 2A, B).

Fig. 2 — The effect of proteolytic enzyme treatments on the culture supernatant of isolates B8.1 (A) and B33.1 B. A supernatant (control), B: supernatant with pepsin, C: supernatant with α-chemotrypsin, D: supernatant with catalase, E: supernatant with trypsin and F: supernatant with proteinase K

Identification of bacteriocin-producing isolates

According to the 16 S rDNA sequence analysis, isolates B7.2, B7.3, B8.1, and B9.1 were identified as E. faecium, while isolates B20.2, B31.2, and B33.1 were identified as E. mundtii. Additionally, isolate B8.2 showed equal similarity to both E. faecium and E. durans species. The 16 S rDNA sequence analysis results for the eight bacteriocin-producing isolates were further verified using Enterococcus genus- and species-specific PCRs. It was determined that all isolates produced 112 bp fragments specific to the Enterococcus genus. Species-specific PCR analysis showed that five of the eight isolates (B7.2, B7.3, B8.1, B8.2, and B9.1) produced 215 bp fragments specific to E. faecium, while the remaining three isolates (B20.2, B31.2, and B33.1) produced 98 bp fragments specific to E. mundtii.

Genotyping of Enterococcus isolates by RAPD-PCR

The genotyping analysis of the isolates using the RAPD-PCR method showed that they had distinct banding patterns from one another (Fig. 3). The isolates were categorized into two distinct clusters, namely A and B. E. mundtii isolates were grouped in cluster A, while E. faecium isolates were grouped in cluster B. Among the E. mundtii isolates, the highest similarity, approximately 76.9%, was detected between the B20.2 and B33.1 strains. Among the E. faecium isolates, the highest similarity, approximately 87.2%, was observed between the B7.2 and B7.3 strains. The similarity percentages among the strains based on the RAPD-PCR band profiles are presented in Supplementary Table S4.

The effect of different enzyme, heat and pH treatments on bacteriocin activity

The enzyme treatments showed that proteinase K, trypsin, α-chymotrypsin, and α-amylase completely removed the antibacterial activity of CFSs. On the other hand, pepsin only slightly reduced this activity. Additionally, lysozyme, lipase, and catalase enzymes did not affect the antibacterial activities of CFSs (Table 3).

Table 3.

The effect of different enzyme, heat, and pH treatments on the activity of bacteriocins produced by Enterococcus strains (AU/mL)

Treament	Bacteriocin producer Enterococcus strains
Treament	B7.2	B7.3	B8.1	B8.2	B9.1	B20.2	B31.2	B33.1
Control	800	800	1600	1600	1600	800	800	800
Enzyme
Proteinase K	0	0	0	0	0	0	0	0
Trypsin	0	0	0	0	0	0	0	0
Pepsin	400	400	800	100	800	400	400	400
α-Chymotrypsin	0	0	0	0	0	0	0	0
Catalase	800	800	1600	1600	1600	800	800	800
Lysozyme	800	800	1600	1600	1600	800	800	800
Lipase	800	800	1600	1600	1600	800	800	800
α-Amylase	0	0	0	0	0	0	0	0
pH
2.0	1600	800	1600	3200	1600	1600	800	1600
3.0	1600	800	1600	3200	1600	1600	800	1600
4.0	1600	800	1600	1600	1600	1600	800	800
5.0	800	800	1600	1600	1600	1600	800	800
6.0	800	800	1600	1600	1600	800	800	800
7.0	800	800	1600	1600	1600	800	800	800
8.0	800	800	1600	1600	800	800	800	800
9.0	800	800	1600	1600	400	800	400	800
10.0	800	800	1600	1600	400	400	400	400
11.0	800	400	800	800	200	200	100	100
Heat
100 °C for 5 min	800	800	1600	1600	1600	800	800	800
100 °C for 10 min	800	800	1600	1600	1600	800	800	800
100 °C for 15 min	800	800	1600	1600	1600	800	800	800
100 °C for 20 min	800	800	1600	1600	1600	400	400	800
121 °C for 15 min	0	0	0	100	200	0	0	0

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The bacteriocins produced by all isolates, except E. mundtii B20.2 and B31.2, were found to be resistant to heat treatment at 100 °C for 5, 10, 15, and 20 min. However, after 20 min at this temperature, the bacteriocins from the B20.2 and B31.2 lost 50% of their activity. Following heat treatment at 121 °C for 15 min, the bacteriocins produced by all isolates, except E. faecium B8.2 and B9.1, completely lost their activity (Table 3).

At acidic pH levels, the antibacterial activity of bacteriocins produced by E. faecium B7.2, B8.2, E. mundtii B20.2, and B33.1 increased by 100%, while the activity of bacteriocins produced by other isolates remained stable. Additionally, it was observed that, with the exception of E. faecium B7.2, whose activity remained stable, the bacteriocins produced by all other isolates partially lost their activity at basic pH levels (Table 3).

Detection of enterocin-encoding genes by PCR, tricine-SDS-PAGE and detection of active protein bands

PCR analysis revealed that E. faecium strains contained both the entA and entB genes, whereas E. mundtii strains contained only the munKS gene (Table 4; Fig. 4).

Table 4.

Isolation source, detected enterocin genes and technological properties of bacteriocin-producing Enterococcus strains

Strains	Source/City	Enterocin gene	Acid production*		Proteolytic activity	Lipolytic activity	Nitrate reductase activity	Enzyme profile
Strains	Source/City		∆pH_{6th hour}	∆pH_{24th hour}		Spirit blue agar / LB agar		Enzyme profile
E. faecium B7.2	Veal/Antalya	entA⁺, entB⁺	1.63 ± 0.036^Ab	2.04 ± 0.070^Aa	-	- / -	-	esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase
E. faecium B7.3	Veal/Antalya	entA⁺, entB⁺	1.58 ± 0.027^Ab	1.91 ± 0.017^Ba	-	- / -	-	esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase
E. faecium B8.1	Lamb meat/ Antalya	entA⁺, entB⁺	1.61 ± 0.020^Ab	1.96 ± 0.015^Ba	-	- / -	-	esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase
E. faecium B8.2	Lamb meat/ Antalya	entA⁺, entB⁺	1.59 ± 0.000^Ab	1.96 ± 0.015^Ba	-	- / -	-	esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase
E. faecium B9.1	Veal/Antalya	entA⁺, entB⁺	1.62 ± 0.012^Ab	1.95 ± 0.040^Ba	-	- / -	-	esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase
E. mundtii B20.2	Veal/Antalya	munKS ⁺	1.08 ± 0.021^Bb	1.71 ± 0.023^Ca	-	- / -	+	esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase
E. mundtii B31.2	Sucuk/ Afyonkarahisar	munKS ⁺	1.09 ± 0.017^Bb	1.66 ± 0.021^CDa	-	- / -	+	esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase
E. mundtii B33.1	Lamb meat/ Isparta	munKS ⁺	1.02 ± 0.015^Bb	1.63 ± 0.001^Da	-	- / -	+	esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol AS-Bl phosphohydrolase

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* Values are expressed as mean ± standard deviation (n = 3). Different uppercase letters indicate significant differences among test organisms at the same time point, whereas different lowercase letters indicate significant differences between time points within the same test organism (P < 0.05)

Fig. 4 — Agarose gel electrophoresis of PCR products amplified using *munKS*- and *CRL35*-specific primers. Line 1: *E. faecium* B7.2, line 2: *E. faecium* B7.3, line 3: *E. faecium* B8.1, line 4: *E. faecium* B8.2, line 5: *E. faecium* B9.1, line 6: *E. mundtii* 20.2, line 7: *E. mundtii* 31.2, line 8: *E. mundtii* 33.1, line 9: *E. mundtii* YB6.30 (positive control for *munKS*⁺), line 10: negative control for *munKS* (water), line M: Genesta™ 100-bp DNA marker (GeneAll Bldg, GA-010, Seoul, Korea), line 11: *E. faecium* B7.2, line 12: *E. faecium* B7.3, line 13: *E. faecium* B8.1, line 14: *E. faecium* B8.2, line 15: *E. faecium* B9.1, line 16: *E. mundtii* 20.2, line 17: *E. mundtii* 31.2, line 18: *E. mundtii* 33.1, line 19: negative control for *CRL35* (water)

Tricine-SDS-PAGE analysis showed that partially purified bacteriocin samples from E. faecium strains B7.2, B7.3, B8.1, B8.2, and B9.1 each exhibited a single active protein band approximately 5.50 kDa in size. Similarly, bacteriocin samples from E. mundtii strains B20.2, B31.2, and B33.1, which had been partially purified, showed a single active protein band around 5.0 kDa (Fig. 5).

Fig. 5 — Tricine-SDS-PAGE profiles and detection of active protein bands of bacteriocins produced by *E. mundtii* strains. Lines 1 and 5: multicolour low range protein ladder (Thermo Scientific, #26628), line 2–4: the approximately 5.0 kDa protein band representing mundticin KS (arrow) produced by E. mundtii B20.2, B31.2, and B33.1 strains, respectively, lines 6–8: inhibition of E. faecium ATCC 51,559 (arrow), embedded in MRS soft agar

Technological properties of bacteriocin producer Enterococcus strains

The acidifying activity of the strains was evaluated by monitoring pH changes (ΔpH) after 6 and 24 h of incubation. The ΔpH values ranged from 1.02 ± 0.012 to 1.63 ± 0.012 after 6 h, and from 1.63 ± 0.018 to 2.04 ± 0.018 after 24 h of incubation (Table 4). Two-way ANOVA revealed significant main effects of strain and incubation time on acidification capacity (P < 0.05), as well as a significant strain × time interaction (P < 0.05). Post hoc comparisons using the LSD test indicated that, at both incubation times, all E. faecium strains exhibited significantly higher acidification capacity than E. mundtii strains (P < 0.05). Minor but statistically significant differences were also observed among E. faecium strains within the same incubation time. Overall, acidification capacity significantly increased from 6 to 24 h for all strains, confirming a pronounced time-dependent effect.

None of the bacteriocin-producing Enterococcus strains were able to hydrolyze casein after 3 days of incubation at 37 °C. Additionally, neither strain showed extracellular lipolytic activity on spirit blue agar or LB agar supplemented with Tween 80 (Table 4).

The results of the API^® ZYM enzyme profiling experiments demonstrated that all Enterococcus strains exhibited activities of esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. Additionally, E. faecium strains produced valine arylamidase and cystine arylamidase, while E. mundtii strains did not. The API^® ZYM test also revealed that none of the strains produced alkaline phosphatase, lipase, trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, or α-fucosidase, or that these enzymes were produced at very low levels (Table 4).

The nitrate reductase activity assay indicated that E. mundtii strains exhibited nitrate reductase activity, whereas E. faecium strains did not (Table 4).

Safety properties of bacteriocin-producing Enterococcus strains

Antibiotic resistance: phenotypical and genotypical determination

All strains were found to be sensitive to ampicillin, penicillin G, vancomycin (with the exception of E. faecium B9.1), tetracycline, doxycycline, minocycline, levofloxacin (with the exception of E. faecium B9.1), norfloxacin, nitrofurantoin (100 µg), chloramphenicol, quinupristin-dalfopristin (with the exception of E. mundtii B33.1), linezolid, gentamicin, and streptomycin. Conversely, most strains exhibited resistance to tigecycline (6/8, 75%) and rifampin (6/8, 75%), followed by teicoplanin (5/8, 62.5%), ciprofloxacin (5/8, 50%), nitrofurantoin (300 µg) (4/8, 50%), imipenem (2/8, 25%), and levofloxacin (1/8, 12.5%). Additionally, all isolates (100%) were found to be intermediate resistance to erythromycin, followed by imipenem (6/8, 75%), nitrofurantoin (300 µg) (1/8, 12.5%), quinupristin-dalfopristin (1/8, 12.5%), and vancomycin (1/8, 12.5%) (Table 5). The MAR index of the strains ranged from 0.00 to 0.26, with a mean value of 0.16. The E. faecium strains exhibited higher MAR indices than the E. mundtii strains, and the E. faecium strains were identified as being multidrug-resistant.

Table 5.

Antibiotic susceptibility and resistance percentages (%) of bacteriocin-producing Enterococcus strains

Antibiotics	Concentration µg/disc	Susceptible		Intermediate		Resistant
Antibiotics	Concentration µg/disc	n^d	%	n	%	n	%
Ampicillin^a	2	8	100.0	0	0.0	0	0.0
Ampicillin^b	10	8	100.0	0	0.0	0	0.0
Imipenem^a	10	0	0.0	6	75.0	2	25.0
Tigecycline^a	15	2	25.0	0	0.0	6	75.0
Penicillin G^b	10^c	8	100.0	0	0.0	0	0.0
Vancomycin^b	30	7	87.5	1	12.5	0	0.0
Teicoplanin^a	30	3	37.5	0	0.0	5	62.5
Erythromycin^b	15	0	0.0	8	100.0	0	0.0
Tetracycline^b	30	8	100.0	0	0.0	0	0.0
Doxycycline^b	30	8	100.0	0	0.0	0	0.0
Minocycline^b	30	8	100.0	0	0.0	0	0.0
Ciprofloxacin^a	5	3	37.5	0	0.0	5	62.5
Levofloxacin^a	5	7	87.5	0	0.0	1	12.5
Norfloxacin^a	10	8	100.0	0	0.0	0	0.0
Nitrofurantoin^a	100	8	100.0	0	0.0	0	0.0
Nitrofurantoin^b	300	3	37.5	1	12.5	4	50.0
Rifampin^b	5	2	25.0	0	0.0	6	75.0
Chloramphenico^b	30	8	100.0	0	0.0	0	0.0
Quinupristin-dalfopristin^a	15	7	87.5	1	12.5	0	0.0
Linezolid^b	30	8	100.0	0	0.0	0	0.0
Gentamycin^a	30	8	100.0	0	0.0	0	0.0
Gentamycin^b	120	8	100.0	0	0.0	0	0.0
Streptomycin^a	300	8	100.0	0	0.0	0	0.0

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^aThe susceptibility or resistance of strains was determined according to the guidelines of EUCAST (2021)

^bThe susceptibility or resistance of strains was determined according to the guidelines of CLSI (2016)

^cPenicillin G U/disc

^dn isolate number

The results of the PCR amplification indicated that the E. faecium B7.2 and B7.3 strains did not harbor any of the antibiotic resistance genes analyzed in this study. In contrast, the remaining six strains were found to possess between one and five antibiotic resistance genes. The highest number of resistance genes was detected in E. faecium B8.2, which contains tetL, aac(6ʹ)-Ie-aph(2ʹʹ)-Ia, aph(3ʹ)-IIIa, ant(4ʹ)-Ia, and ant(6ʹ)-Ia (Table 6). The most frequently observed resistance genes were tetL, aph(3ʹ)-IIIa, and ant(4ʹ)-Ia, detected in 50% (4/8) of the strains. These were followed by ant(6ʹ)-Ia (3/8, 37.5%) and aac(6ʹ)-Ie-aph(2ʹʹ)-Ia (2/8, 25%).

Table 6.

Antibiotic resistance patterns, antibiotic resistance genes, hemolytic and gelatinase activities, virulence factor genes, biogenic amin production, and amino acid decarboxylase genes in bacteriocin-producing Enterococcus strains

Strain	Antibiotic resistance^a	MAR^c index	Antibiotic resistance genes	Hemolytic/gelatinase activities	Virulence factor genes	Biogenic amin production/ decarboxylase gene
E. faecium B7.2	CIP, F^b, IMP, RD, TEC, TGC	0.26	-	γ / negative	efaA_fm,	Tyramine positive / tdc
E. faecium B7.3	CIP, F^b, IMP, RD, TEC, TGC	0.26	-	γ / negative	efaA_fm,	Tyramine positive / tdc
E. faecium B8.1	CIP, F^b, RD, TEC, TGC	0.22	aac(6ʹ)-Ie-aph(2ʹʹ)-Ia, aph(3ʹ)-IIIa, ant(4ʹ)-Ia	γ / negative	efaA_fm, acm	Tyramine positive / tdc
E. faecium B8.2	CIP, F^b, RD, TEC, TGC	0.22	aac(6ʹ)-Ie-aph(2ʹʹ)-Ia, aph(3ʹ)-IIIa, ant(4ʹ)-Ia, ant(6ʹ)-Ia, tetL	γ / negative	efaA_fm, acm	Tyramine positive / tdc
E. faecium B9.1	CIP, LEV, RD, TEC, TGC	0.22	ant(6ʹ)-Ia, tetL	γ / negative	efaA_fm, acm	Tyramine positive / tdc
E. mundtii B20.2	RD	0.04	aph(3ʹ)-IIIa, ant(4ʹ)-Ia	γ / negative	acm	Tyramine weak positive / tdc
E. mundtii B31.2	-	-	tetL	γ / negative	acm	Tyramine weak positive / tdc
E. mundtii B33.1	TGC	0.04	aph(3ʹ)-IIIa, ant(4ʹ)-Ia, ant(6ʹ)-Ia, tetL	γ / negative	-	Tyramine weak positive / tdc

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^aCIP Ciprofloxacin, F Nitrofurantoin, IMP Imipenem, LEV Levofloxacin, RD Rifampin, TEC Teicoplanin, TGC Tigecycline

^bNitrofurantoin 300 µg/disc

^cMAR Multiple antibiotic resistance index

Virulence factors: phenotypical and genotypical determination

It has been established that all strains exhibit γ-hemolytic activity. Similarly, none of the strains demonstrated gelatinase activity (Table 6).

The results of the PCR analysis revealed that bacteriocin-producing strains exhibited a low number of virulence factor genes. Specifically, E. mundtii B33.1 did not possess any virulence factor genes. No detection of the gelE, efaA_fs, esp_fm, esp_fs, cpd, cob, ccf, cad, ace, agg, cylM, cylB, cylA, or hyl genes was observed in any of the strains. In contrast, efaA_fm and acm were the most commonly identified virulence factor genes, being present in 62.5% of the strains. Both efaA_fm and acm were detected in E. faecium B8.1, B8.2, and B9.1. Additionally, efaA_fm was found in E. faecium B7.2 and B7.3, whereas acm was only present in E. mundtii B20.2 and B31.2 (Table 6).

Biogenic amine production: phenotypical and genotypical determination

The results of the decarboxylase activity test indicated that none of the strains were able to decarboxylate histidine, lysine, or ornithine. In contrast, the E. faecium strains B7.2, B7.3, B8.1, B8.2, and B9.1 exhibited decarboxylation activity for tyrosine, whereas the E. mundtii strains B20.2, B31.2, and B33.1 demonstrated weak decarboxylation activity (Table 6).

The PCR analysis results indicated that none of the strains amplified products specific to the hdc, ldc, or odc genes. However, all strains produced a 825-bp amplicon specific to the tdc gene (Fig. 6). These PCR findings are consistent with the results obtained from phenotypic testing.

Fig. 6 — Agarose gel electrophoresis of PCR products amplified using *tdc* primers. Line 1: E. faecium B7.2, line 2: E. faecium B7.3, line 3: E. faecium B8.1, line 4: E. faecium B8.2, line 5: E. faecium B9.1, line 6: E. mundtii 20.2, line 7: E. mundtii 31.2, line 8: E. mundtii 33.1, line 9: E. faecalis NYE54 (positive control), line 10: negative control (water), line M: O’GeneRuler™ 100 bp DNA ladder (Thermo Fisher Scientific, #SM1153, USA), line 11: negative control (water), line 12: E. faecalis NYE54 (positive control)

Discussion

The most important step in screening for bacteriocin-producing strains is selecting those that form a zone of inhibition against the indicator bacteria. Consequently, the presence of a clear, sharply defined inhibition zone around the colony or well is considered strong preliminary evidence that the tested strain may produce a bacteriocin [75–77], although such inhibition may also result from other antimicrobial metabolites produced by LAB. During the screening of raw meat and meat products for bacteriocin-producing LAB strains, a total of 18 isolates were identified as potential bacteriocin producers. Notably, calf samples yielded the highest number of bacteriocin-producing isolates, followed by sucuk and mutton, whereas no antimicrobial activity–producing LAB were recovered from pastırma samples. All isolates were found to be Gram-positive and catalase-negative, confirming their presumptive classification as LAB. Consistent with our findings, several studies have reported the presence of bacteriocin-producing LAB in raw meat and meat products, particularly fermented sausages, in different countries, including Brazil [78], Türkiye [26], Italy [5], Germany [79], and Portugal [80, 81]. In contrast to our results, Güllüce et al. [82] reported that LAB isolates obtained from pastırma exhibited significant bacteriocinogenic potential, identifying one Lactobacillus plantarum, nine L. plantarum subsp. plantarum, and one Pediococcus pentosaceus.

The loss of activity caused by proteolytic enzyme treatment supports the idea that the antibacterial compound is protein-based. Bacteriocins, which are protein-based antibacterial substances produced by bacteria, are known to lose part or all of their activity when treated with proteolytic enzymes due to their protein nature. Additionaly, the unaffected antibacterial activity from the catalase treatment suggests that it is not caused by H₂O₂ [25, 44, 77, 83, 84]. Based on these findings, the isolates B7.2, B7.3, B8.1, B8.2, B9.1, B20.2, B31.2, and B33.1 were identified as bacteriocin-producing LAB. The identification of these bacteriocin-producing isolates was carried out through 16 S rDNA sequence analysis and further confirmed by PCR-based molecular methods targeting the elongation factor EF-Tu (tuf) and manganese-dependent superoxide dismutase (sodA) genes, which enabled genus- and species-specific identification of enterococci, respectively. The tuf gene is involved in peptide chain synthesis and contains a conserved region that reliably detects the Enterococcus genus [85]. Additionally, sodA is regarded as one of the most effective genes for identifying enterococci at the species level [38]. Based on the identification analysis, five of the eight isolates were identified as E. faecium, while the remaining three isolates were identified as E. mundtii. Enterococci, which are part of the normal microbiota in the gastrointestinal tracts of animals, can contaminate meat during slaughter. As a result, Enterococcus strains are commonly found in meat and meat products [28, 53, 86–88]. Consistent with our findings, Enterococcus species exhibiting antibacterial activity have been isolated from various meat products, including sucuk, a dry-fermented sausage from Türkiye [26]; Lombo [80] and Alheira [81] sausages from Portugal; pastırma, a dry-cured meat from Türkiye [88]; Gueddid, a dry-fermented meat from Tunisia [89]; and Sokobanja sausage from Serbia [90]. Previous studies indicate that E. faecium and E. faecalis species are more frequently bacteriocin producers compared to other Enterococcus species found in food sources. Fewer studies have reported bacteriocins produced by E. mundtii strains isolated from sucuk [26], goat and sheep colostrum [77], traditional Turkish cheeses [25], and plant materials [43, 91–93].

The genotyping analysis of bacteriocin-producing Enterococcus strains, conducted using the RAPD-PCR method, revealed that the E. faecium and E. mundtii isolates were grouped into two distinct clusters, each displaying unique banding patterns. Similar to our findings, RAPD-PCR has been successfully used to genotype bacteriocin-producing enterococci in colostrum samples [77], traditional cheeses [25], and fresh shrimp [94].

Enzyme assays demonstrated that the proteolytic enzymes—proteinase K, trypsin, and α-chymotrypsin—completely inactivated the antibacterial activity of bacteriocins produced by Enterococcus strains, while pepsin led to partial inactivation, consistent with previous reports on enterococcal bacteriocins [25, 77]. Additionally, treatment with α-amylase resulted in the complete elimination of the bacteriocins’ antibacterial activity (Table 3). Sensitivity to α-amylase has previously been reported for bacteriocins described as carbohydrate-associated or glycoprotein-like in enterococci [25, 77, 83, 95–97]. Gök Charyyev et al. [83] indicated that the activity of a presumptive enterocin B produced by E. faecium YT52 was partially inactivated following α-amylase treatment. More recently, Mancini et al. [97] reported that an enterocin B produced by E. faecium RM12 exhibited sensitivity to proteolytic enzymes and a partial reduction in activity after α-amylase treatment, suggesting the possible involvement of carbohydrate-containing components in its antibacterial activity. In contrast, only a limited number of studies have investigated the effect of α-amylase on the antibacterial activity of mundticins [25, 77, 98]. Todorov et al. [98] reported that no change in antimicrobial activity was observed after α-amylase treatment, indicating that the peptide ST4V produced by E. mundtii ST4V was not glycosylated. These findings suggest that, unlike some enterocins, mundticins are generally non-glycosylated peptides, and that any observed α-amylase sensitivity may be strain-dependent or influenced by methodological factors, such as the use of crude cell-free supernatants rather than purified bacteriocin preparations. Further structural and purification-based investigations are therefore required to clarify this observation.

After heat treatment at 100 °C for 5, 10, and 15 min, the bacteriocins produced by all isolates retained their activity. However, after heat treatment at 100 °C for 20 min and 121 °C for 15 min, the bacteriocins produced by the isolates generally lost activity to varying degrees. Similarly, enterocins have been reported to be heat-stable, maintaining their activity at 100 °C [25, 47, 77, 83, 99, 100]. Consistent with our findings, previous studies have shown that the activity of enterocins increases at acidic pH levels due to enhanced solubility, whereas their activity decreases under alkaline conditions [77, 83]. These findings indicate that bacteriocins produced by Enterococcus strains may be applicable in food products across a broad range of pH levels and processing conditions involving pasteurization temperatures. The presence of multiple enterocin genes in E. faecium strains has been well-documented in several studies [24, 25, 46, 47, 77, 83, 100]. Consistent with these findings, PCR analysis revealed that E. faecium strains harbored both the entA and entB genes (Table 4). These genes are typically found together in enterococci [24, 46, 77, 83, 100]. The entA and entB genes are regulated by the same system, and the corresponding bacteriocins are secreted from the cell through the same ABC transporter [101]. On the other hand, E. mundtii strains were found to contain only the munKS gene (Table 4; Fig. 4), as previously reported by Öztürk et al. [77]. To date, only a limited number of studies have reported the production of bacteriocins by certain E. mundtii strains, including mundticin QU2 [43], mundticin [91], mundticin L [92], mundticin KS [25, 26, 77, 93], and mundticin CRL35 [102]. Notably, mundticin-producing Enterococcus strains are commonly isolated from plant sources [43, 84–86]. In this study, potential mundticin KS-producing E. mundtii strains were identified in raw meat (B20.2 and B33.1) and sucuk (B31.2). Similarly, Altınkaynak and Tuncer [26] isolated the mundticin KS-producing E. mundtii YB6.30 strain from sucuk. Additionally, Öztürk et al. [77] and Akpınar-Kankaya [25] reported mundticin KS-producing E. mundtii strains in small livestock colostrums and traditional cheeses, respectively.

Tricine-SDS-PAGE is a widely used technique for effectively separating proteins with molecular weights under 30 kDa [48]. Consistent with our findings, earlier research has indicated that the molecular weights of enterocins are less than 10 kDa [26, 83, 103]. The tricine-SDS-PAGE analysis of E. faecium strains B7.2, B7.3, B8.1, B8.2, and B9.1 revealed that the sizes of the active protein bands (~ 5.50 kDa) were close to the theoretical molecular weights calculated based on the amino acid sequences of enterocin B. These results suggest that E. faecium strains are likely enterocin B producers. Enterocin B, a group IIc-member bacteriocin that lacks the YGNGVXC consensus motif in its N-terminal region, consists of 53 amino acid residues and has a molecular weight of 5463 Da. It exhibits antibacterial activity against L. monocytogenes [104, 105]. When comparing the active protein band patterns and enterocin gene contents of E. faecium strains, it is hypothesized that the entA gene is either not expressed in these strains (silent) or expressed at such low levels that it cannot be detected by tricine-SDS-PAGE. Previous research has also shown that some Enterococcus strains harbor silent enterocin genes [77, 83, 106]. Tricine-SDS-PAGE analysis of bacteriocins produced by E. mundtii strains revealed an active protein band (~ 5.0 kDa), consistent in size with mundticin KS (Fig. 5). These findings confirmed the phenotypic expression of the munKS gene in E. mundtii strains B20.2, B31.2, and B33.1, as identified by PCR analysis. Thus, these strains were identified as potential mundticin KS producers. Kawamoto et al. [93] reported that purified mundticin KS from E. mundtii NFRI 7393 yielded a single active protein band (3.4–6.5 kDa) in tricine-SDS-PAGE. Similarly, Öztürk et al. [77] reported a single active peptide band (~ 4.96 kDa) for partially purified mundticin KS from eleven E. mundtii strains isolated from goat and sheep colostrum.

The acidification ability of LAB is a key criterion for selecting strains as starter cultures in fermented food production [8, 107]. Organic acids not only inhibit the growth of spoilage and pathogenic microorganisms but also play a crucial role in shaping the organoleptic properties of the final product [107]. While Enterococcus species are generally recognized for their slow to moderate acid production in milk-based environments, several studies have demonstrated that their acidification potential varies depending on the strain, incubation period, and culture medium [106–109]. Given their acid production profile presented in Table 4, bacteriocin-producing Enterococcus strains, isolated from raw meat and meat products, may have potential as starter or adjunct cultures for use in fermented food production. Enterococcus strains are generally known to exhibit low or absent extracellular proteolytic and lipolytic activity [44, 53, 77], as confirmed in the present study (Table 4). Ben Belgacem et al. [44] assessed the proteolytic activity of 24 enterococcal strains with antibacterial properties, isolated from the Tunisian fermented meat product Gueddid, and found no evidence of proteolytic activity. Similarly, Landeta et al. [53] reported that none of the 19 E. faecium isolates from Spanish sausages hydrolyzed casein on calcium caseinate agar. Furthermore, Öztürk et al. [77] indicated that enterocin-producing 11 E. mundtii and two E. faecium strains, isolated from goat and sheep colostrum, did not hydrolyze casein on calcium caseinate agar. Similar to our results, Kasap and Tuncer [109] investigated the lipolytic activity of the mundticin KS-producing E. mundtii YB6.30 strain, isolated from sucuk, in four different media (spirit blue agar, tributyrin agar, and MRS agar containing 1% (v/v) Tween 80 or Tween 20) and found no lipolytic activity. Furthermore, Aspri et al. [110] reported that none of the enterococcal isolates from donkey milk showed lipolytic activity on tributyrin agar. Moreover, more recently, Öztürk et al. [77] and Akpınar-Kankaya [25] reported that none of the bacteriocinogenic enterococci strains isolated from colostrum and cheese samples exhibited lipolytic activity, respectively. On the other hand, there are studies by other researchers reporting that enterococcal isolates show lipolytic activity. In these studies, it was generally found that enterococci exhibited low levels of lipolytic activity [107, 108, 111]. Although lipolytic activity is a desirable feature in strains selected as starter cultures, high lipolytic activity may negatively affect the organoleptic properties of the final product [112]. For this reason, the lack of lipolytic activity in enterocin-producing strains is not considered a disadvantage for their use as starter cultures. Consistent with our findings, previous studies have reported that species within the Enterococcus genus generally exhibit esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase activities [77, 113–116]. However, some studies have reported that various Enterococcus strains also produce alkaline phosphatase, valine arylamidase, cystine arylamidase, α-chymotrypsin, β-galactosidase, and β-glucosidase [77, 115, 117, 118]. Various studies have demonstrated that enterococcal strains exhibit significantly higher esterolytic activity [22, 44, 77, 113–119], a finding corroborated by the present study. Esterase and esterase lipase activities play a crucial role in the conversion of fats into short-chain fatty acids and glycerol [119, 120]. These enzymes contribute to the increase fatty acids in cheese [119]. Additionally, they are important for treating metabolic disorders such as hypercholesterolemia and hyperlipidemia, as well as for formulating nutritional preparations for newborns and seniors [120]. In bacteriocin-producing Enterococcus strains, protease activity (trypsin and α-chymotrypsin) was absent, while peptidase activity (leucine arylamidase in E. mundtii and both leucine and valine arylamidase in E. faecium strains) was elevated. This characteristic makes bacteriocin-producing Enterococcus strains advantageous as starter or adjunct cultures in cheese production, as low protease and high peptidase activity contribute to reduced bitterness and improved cheese texture and structure during production and ripening [119]. Additionally, acid phosphatase and phosphohydrolase production were detected in all strains. These enzymes play a crucial role in phosphopeptide breakdown during cheese ripening [113, 119]. The absence of β-galactosidase activity in bacteriocin-producing Enterococcus strains may be due to the predominance of phospho-β-galactosidase as the primary glycosidase in these bacteria [119]. Notably, the absence of α-galactosidase, β-glucuronidase, α/β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase activities in Enterococcus strains is advantageous. These enzymes can convert various promutagens and procarcinogens into their mutagenic and carcinogenic forms [115]. The nitrate reductase activity test revealed that E. mundtii strains exhibited nitrate reductase activity, whereas E. faecium strains did not (Table 4). Similarly, previous studies by Landeta et al. [53] and Kasap and Tuncer [109] reported that enterococci isolated from fermented meat products also displayed nitrate reductase activity. Nitrite, a widely used additive in meat products, has been employed in the food industry for many years to inhibit pathogenic microorganisms, particularly Clostridium botulinum, prevent oxidative rancidity, and enhance the characteristic color, flavor, and texture of cured meats [121]. Although Gram-positive, catalase-positive cocci (GCC) are primarily responsible for color formation in fermented sausages, LAB with nitrate reductase activity also contribute to this process. While LAB exhibit lower nitrate reductase activity than GCC, this activity plays a direct role in the formation of nitrosomyoglobin, a key pigment in fermented meat products. Therefore, studying nitrate reductase activity in LAB is essential [53, 121]. Enterocin-producing Enterococcus mundtii strains, which demonstrate nitrate reductase activity, are particularly advantageous as starter or adjunct cultures in fermented meat production.

Enterococci are naturally resistant to β-lactam antibiotics, such as penicillin, ampicillin, piperacillin, and imipenem [122]. However, all bacteriocin-producing Enterococcus strains have been found to be sensitive to ampicillin and penicillin. In line with these findings, several studies have reported that all foodborne enterococci are sensitive to ampicillin [28, 53, 123], with approximately 90% showing sensitivity to penicillin [28, 65, 67, 123]. All Enterococcus strains in the present study were found to be sensitive to tetracycline group antibiotics (tetracycline, doxycycline, minocycline), with the exception of tigecycline. These results are consistent with the findings of Aspri et al. [110], Zheng et al. [124], and Zommiti et al. [125]. Linezolid is an important therapeutic agent for treating infections caused by Gram-positive pathogens, particularly vancomycin-resistant enterococci [126]. Therefore, the sensitivity of all bacteriocin-producing Enterococcus strains to linezolid is an advantage. Aminoglycosides, commonly used in combination with glycopeptides and β-lactams for treating enterococcal infections [65, 127]. Severel studies have reported high aminoglycoside resistance in Enterococcus strains isolated from animal foods [28, 66, 123, 128, 129]. However, in the present study, all Enterococcus strains were found to be sensitive to both low and high concentrations of aminoglycosides, including gentamicin and streptomycin. The susceptibility of Enterococcus to the glycopeptide antibiotic vancomycin is a key criterion for their safety evaluation [65, 125]. Notably, with the exception of E. faecium B9.1, all bacteriocin-producing Enterococcus strains in this study were sensitive to vancomycin. The MAR index of E. mundtii strains was relatively low, ranging from 0.00 to 0.04, whereas E. faecium strains exhibited a MAR index exceeding 0.20, with all identified as MDR. Bacteria resistant to multiple antibiotics pose a significant public health risk by limiting treatment options and exacerbating antimicrobial resistance [130]. Consequently, the use of bacteriocin-producing E. faecium strains as live cultures in food production may present potential health risks to consumers. A MAR index greater than 0.2 indicates that the tested isolates likely originated from high-risk contamination sources with frequent antibiotic use [131].

Tetracycline resistance in enterococci is primarily attributed to the tetM and tetL genes, which encode a ribosomal protection protein and an ATP-dependent efflux protein associated with the membrane, respectively [122, 123, 132]. However, although the tetL gene was detected in E. faecium B8.2 and B9.1, as well as E. mundtii B31.2 and B33.1, all isolates remained phenotypically susceptible to tetracycline. This discrepancy suggests a lack of gene expression. Similarly, Cauwerts et al. [133] found that among three phenotypically tetracycline-sensitive E. faecium strains, two harbored the tetM gene, while one carried the tetL gene. Demirgül and Tuncer [28] also reported that the phenotypically tetracycline-sensitive E. faecium FYE41 contained both tetM and tetL genes. Likewise, Gök Charyyev et al. [83] observed that although the bacteriocin-producing E. faecium YT52 carried both tetM and tetL, it remained phenotypically sensitive to tetracycline. As a result of PCR trials, the aph(3ʹ)-IIIa and ant(4ʹ)-Ia genes were detected in E. faecium B8.1, B8.2, and E. mundtii B20.2, B33.1 strains, despite their phenotypic susceptibility to gentamicin and streptomycin. Additionally, the ant(6ʹ)-Ia gene was identified in E. faecium B8.2 and B9.1, as well as in E. mundtii B33.1, while the aac(6ʹ)-Ie-aph(2ʹʹ)-Ia gene was detected in E. faecium B8.1 and B8.2. In enterococci, high-level resistance to gentamicin and kanamycin is mediated by AAC(6ʹ)-APH(2ʹʹ), a bifunctional aminoglycoside-modifying enzyme (AME) encoded by the aac(6ʹ)-Ie-aph(2ʹʹ)-Ia gene. High-level gentamicin resistance is also associated with the aph(2ʹʹ)-Ib, aph(2ʹʹ)-Ic, and aph(2ʹʹ)-Id genes, which encode monofunctional AMEs. Additionally, the aph(3ʹ)-IIIa, ant(4ʹ)-Ia, and ant(6ʹ)-Ia genes, which encode AMEs, are responsible for resistance against various aminoglycoside antibiotics other than gentamicin [64, 66, 67, 134]. The presence of AME genes in eight bacteriocin-producing Enterococcus strains, despite their susceptibility to gentamicin and streptomycin, suggests potential resistance to other aminoglycosides. Additionally, aminoglycoside resistance genes in phenotypically susceptible strains may result from the amplification of suppressed resistance genes or reduced enzymatic activity of AMEs [135]. Previous studies have also documented that certain S. aureus [136] and E. faecalis strains carry the aac(6ʹ)-Ie-aph(2ʹʹ)-Ia gene while remaining sensitive to gentamicin [137, 138]. Macrolide resistance in enterococci arises through various mechanisms, with the most commonly observed being resistance mediated by erm genes. These genes induce methylation of the 23 S rRNA subunit, thereby preventing macrolides from binding to their target site on the ribosome. While multiple erm genes are present in enterococci, ermB is the most frequently encountered [122]. Notably, bacteriocin-producing Enterococcus strains that exhibited phenotypic intermediate resistance to erythromycin did not harbor the ermA, ermB, or ermC genes. Similarly, other studies have reported that erythromycin-resistant Enterococcus strains also lack these genes [19, 28, 139]. These findings suggest that the intermediate erythromycin resistance observed in bacteriocin-producing Enterococcus strains may result from mechanisms other than ermA, ermB, or ermC. PCR assays designed to detect vancomycin resistance genes revealed that none of the bacteriocin-producing Enterococcus strains harbored the vanA, vanB, vanC1, vanC2, vanC3, vanD, vanE, or vanG resistance genes. These findings are consistent with those of Geniş et al. [19], who reported that none of the enterocin-producing E. mundtii and E. faecium strains isolated from sheep and goat colostrum contained these vancomycin resistance genes. Similarly, Ben Braïek et al. [94] found that eight bacteriocin-producing E. lactis strains, isolated from fresh shrimp, were devoid of the vanA or vanB genes. Moreover, Zommiti et al. [125] reported the absence of vanA or vanB genes in five bacteriocin-producing E. faecium strains isolated from Ossban, a traditional Tunisian dry-fermented meat. Vancomycin is a glycopeptide antibiotic primarily used as a last-resort treatment for infections caused by multidrug-resistant enterococci [65, 140]. Therefore, the absence of transferable vancomycin resistance genes in bacteriocin-producing Enterococcus strains, coupled with their sensitivity to vancomycin, is a crucial factor in assessing the safety of these strains for use as live cultures in the food industry.

Hemolysin/cytolysin, a bacterial toxin, is one of the most studied virulence factors secreted by Enterococcus strains. Hemolytic activity can be encoded by either plasmid or chromosomal DNA and plays a significant role in increasing the severity of human infections [17, 22]. The use of enterococcal strains exhibiting β-hemolytic activity as starter or probiotic culture in food production is not recommended [141]. Therefore, bacteriocin-producing Enterococcus strains that exhibit γ-hemolytic activity are considered advantageous for use as live cultures. Consistent with our findings, previous studies have reported non-hemolytic bacteriocinogenic enterococci isolated from various fermented foods, such as sucuk [109], boza [83], and cheeses [25, 100, 142]. Gelatinase, a zinc-containing extracellular metalloendopeptidase, hydrolyzes bioactive peptides, including gelatin, elastin, collagen, hemoglobin, and pheromone-binding proteins. Its ability to degrade peptides promotes microbial invasion in host tissues, supporting bacterial survival and inflicting direct and indirect damage [17, 22]. Therefore, bacteriocin-producing Enterococcus strains lacking gelatinase activity are advantageous for use as live cultures. Similar to our findings, previous studies have reported the absence of gelatinase activity in foodborne enterococcal isolates with antibacterial activity from sucuk [109], doenjang [143], and traditional Turkish cheeses [25, 142, 144]. However, some studies have reported gelatinase activity in enterococcal isolates from animal-derived foods [86, 142, 145, 146], contrasting these findings.

Effector molecules that enhance the disease-causing potential of microorganisms are called virulence factors. The virulence factors of Enterococcus species play a key role in pathogenicity. Studies indicated that virulence factors are less prevalent in foodborne enterococci than in clinically derived strains [13, 147]. In the present study, among the eight bacteriocin-producing Enterococcus strains, only E. mundtii B33.1 lacks virulence factor genes. Similarly, previous studies have reported that Enterococcus strains isolated from food do not contain virulence factor genes [19, 25, 94, 109]. Cytolysin/hemolysin, one of the best-characterized virulence factors in enterococci, induces hemolysis in erythrocytes from rabbits, humans, horses, and cattle [17, 148]. The absence of the cylM, cylB, and cylA genes in bacteriocin-producing Enterococcus strains supports the phenotypic test results, which showed no hemolytic activity on sheep blood agar. This finding is consistent with previous reports on bacteriocinogenic food-derived Enterococcus strains [19, 25, 83, 109]. In contrast, Anagnostopoulos et al. [107] found that all 64 E. faecium strains isolated from olives exhibited phenotypic γ-hemolytic activity. However, they detected the cylA gene in only two strains (3.1%) and attributed this discrepancy to the possibility that cylA functions as a silent gene. The gelE gene encodes gelatinase, a zinc-dependent metalloendopeptidase (~ 30 kDa) [17]. In the present study, the gelE gene was not detected in bacteriocin-producing Enterococcus strains that were phenotypically determined to be gelatinase-negative, as previously reported by Geniş et al. [19], Gök Charyyev et al. [83], and Akpınar Kankaya [25]. Additionally, the endocarditis antigen efaA, which is encoded by efaA_fs in E. faecalis strains and efaA_fm in E. faecium strains, plays a crucial role in the adherence of enterococci to host cells. It has also been suggested that the efaA antigen contributes to adhesion in endocarditis and influences pathogenicity in animal models [17, 22]. While studies have demonstrated the role of efaA_fs in pathogenicity in animal models, the function of efaA_fm remains unclear [44, 55, 70]. Given its potential role in pathogenicity, the detection of the efaA_fm gene in bacteriocin-producing E. faecium strains (B7.2, B7.3, B8.1, B8.2, and B9.1) raises concerns about their suitability as live cultures in food production. Sex pheromones, small hydrophobic peptides (7–8 amino acids long) encoded on chromosomal DNA, facilitate the conjugative transfer of plasmid DNA among Enterococcus strains. However, they are generally not considered virulence factors in Enterococcus strains [17]. Nonetheless, sex pheromone production is significant, as it facilitates the dissemination of virulence factor genes and antibiotic resistance genes among Enterococcus strains via pheromone-responsive conjugative plasmids [22]. Therefore, the absence of sex pheromone genes in the enterocin-producing Enterococcus strains isolated in this study is considered advantageous for their potential application in food production processes.

Biogenic amines are toxic compounds typically formed by the microbial decarboxylation of specific free amino acids in various foods, including meat, fish, cheese, beer, and wine [22, 149–151]. The lack of dexarboxylase activity for histidine, lysine, and ornithine in bacteriocin-producing Enterococcus strains is advantageous. In contrast, tyrosine dexarboxylation was detected in all strains. Consistent with our findings, several studies indicate that tyramine is the predominant biogenic amine produced by enterococci [19, 53, 151]. PCR analysis corroborated the phenotypic test results. Among the eight bacteriocin-producing Enterococcus strains, E. mundtii B20.2, B31.2, and B33.1 are particularly notable for their potential application as live cultures in the food industry, owing to their weak tyrosine decarboxylation activity and inability to decarboxylate histidine, ornithine, and lysine. Small amounts of biogenic amines are detoxified in the intestine by monoamine and diamine oxidases, producing less physiologically active metabolites [149, 152]. However, further studies are needed to quantify tyramine production in bacteriocin-producing E. mundtii and E. faecium to evaluate their safety in food applications.

Although this study provides a comprehensive evaluation of bacteriocinogenic Enterococcus strains isolated from raw meat and traditional meat products, several limitations should be considered. The isolates were obtained from a limited number of samples collected in selected provinces of Türkiye and may not reflect the overall diversity of Enterococcus populations in meat products. Safety assessment was based on phenotypic analyses and PCR detection of selected genes; whole-genome sequencing would allow a more complete evaluation of resistance determinants, virulence potential, and mobile genetic elements. In addition, technological performance was assessed under laboratory conditions, and biogenic amine formation was not quantified in food matrices despite the detection of the tdc gene. Further genome-based and in situ studies are therefore required.

Conclusion

A total of eight bacteriocin-producing Enterococcus strains were isolated from raw meat and meat products. PCR-based molecular analysis identified five strains as E. faecium and three as E. mundtii. RAPD-PCR analysis revealed genetic dissimilarity among the isolates. The bacteriocins exhibited antimicrobial activity over a broad pH range and demonstrated heat resistance. In E. faecium strains, both entA and entB genes were detected, whereas only the munKS gene was identified in E. mundtii strains. Based on tricine-SDS-PAGE results and gene content analysis, E. faecium strains were classified as likely enterocin B producers, while E. mundtii strains were classified as likely mundticin KS producers. The technological properties of these bacteriocin-producing strains indicate their potential for inclusion in starter culture formulations for fermented foods such as sausage and cheese. Antibiotic susceptibility testing showed that the strains were generally susceptible to clinically relevant antibiotics. Safety evaluation assays suggested that E. mundtii B33.1 appears to be a promising candidate for use as a starter or adjunct culture, pending further genomic and in situ validation. Although this strain did not harbor the investigated virulence genes and did not exhibit hemolytic or gelatinase activity, these findings represent only a preliminary safety assessment. Targeted PCR screening is limited to known determinants and cannot exclude uncharacterized virulence factors. Therefore, whole-genome sequencing is required before any industrial or probiotic application. In contrast, the remaining seven strains may pose potential health risks if applied as live cultures in food production. Nonetheless, purified bacteriocins derived from these strains may offer promising applications in food preservation.

Supplementary Information

Supplementary Material 1.^{(35KB, doc)}

Supplementary Material 2.^{(109KB, doc)}

Supplementary Material 3.^{(39.5KB, doc)}

Supplementary Material 4.^{(10.1KB, pdf)}

Acknowledgements

The authors gratefully acknowledge Prof. Dr. Özgür KOŞKAN (Isparta University of Applied Sciences, Department of Biometry and Genetics, Isparta, Türkiye) for providing support with the statistical analysis of the data.

Abbreviations

CFS: Cell-free supernatant
GCC: Gram-positive catalase-positive cocci
GRAS: Generally Recognized as Safe
EFSA: The European Food Safety Authority
LAB: Lactic acid bacteria
MAR: Multiple antibiotic resistance
MRS: de Man Rogosa and Sharpe
PCR: Polymerase chain reaction
RAPD-PCR: Random amplified polymorphic DNA
Tricine-SDS-PAGE: Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis
QPS: The Qualified Presumption of Safety

Authors’ contributions

Burak Geniş: Conceptualization, investigation, writing the original draft. Yasin Tuncer: Conceptualization, investigation, resources, writing – review & editing, project administration, and funding acquisition. All authors read and approved the manuscript.

Funding

We express our gratitude to the Scientific Research Project Committee of Süleyman Demirel University (Isparta, Türkiye) for its financial assistance under project number FDK 2019–7345. Furthermore, the Council of Higher Education (CoHE) of Türkiye provided author Burak Geniş with funding through the CoHE 100/2000 Ph.D. scholarship program in the field of food biotechnology.

Data availability

The data that support the findings of this study are available from the corresponding author (yasintuncer@sdu.edu.tr) upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1.^{(35KB, doc)}

Supplementary Material 2.^{(109KB, doc)}

Supplementary Material 3.^{(39.5KB, doc)}

Supplementary Material 4.^{(10.1KB, pdf)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author (yasintuncer@sdu.edu.tr) upon reasonable request.

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PERMALINK

Technological properties and safety evaluation of bacteriocinogenic Enterococcus strains isolated from raw meat and traditional meat products in Türkiye

Burak Geniş

Yasin Tuncer

Abstract

Objective

Methods

Results

Conclusion

Supplementary Information

Background

Materials and methods

Meat and meat product samples

Screening of antimicrobial activity of presumptive LAB isolates

Antibacterial activity spectrum of presumptive bacteriocin-producing LAB and detection of the antibacterial substance’s susceptibility to proteolytic enzymes

Table 1.

Table 2.

Identification of bacteriocin-producing isolates

Genotyping of Enterococcus isolates by RAPD-PCR

The effect of different enzyme, heat and pH treatments on bacteriocin activity

Detection of enterocin-encoding genes by PCR

Purification of bacteriocins, tricine-SDS-PAGE and detection of active protein bands

Technological properties of bacteriocin-producing Enterococcus strains

Acid production

Proteolytic and lipolytic activities

Enzyme profiles

Nitrate reductase activity

Safety properties of bacteriocin-producing Enterococcus strains

Antibiotic resistance: phenotypical and genotypical determination

Virulence factors: phenotypical and genotypical determination

Biogenic amine production: phenotypical and genotypical determination

Statistical analysis

Results

Screening of presumptive bacteriocin-producing isolates

Antibacterial activity spectrum of LAB and detection of the antibacterial substance’s susceptibility to proteolytic enzymes

Fig. 1.

Fig. 2.

Identification of bacteriocin-producing isolates

Genotyping of Enterococcus isolates by RAPD-PCR

Fig. 3.

The effect of different enzyme, heat and pH treatments on bacteriocin activity

Table 3.

Detection of enterocin-encoding genes by PCR, tricine-SDS-PAGE and detection of active protein bands

Table 4.

Fig. 4.

Fig. 5.

Technological properties of bacteriocin producer Enterococcus strains

Safety properties of bacteriocin-producing Enterococcus strains

Antibiotic resistance: phenotypical and genotypical determination

Table 5.

Table 6.

Virulence factors: phenotypical and genotypical determination

Biogenic amine production: phenotypical and genotypical determination

Fig. 6.

Discussion

Conclusion

Supplementary Information

Acknowledgements

Abbreviations

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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