Lactic Acid Bacteria from Traditional Fermented Milk: Antimicrobial Potential Against Foodborne Pathogens

Abstract

Lactic acid bacteria (LAB) are increasingly recognized for their role in food biopreservation due to their ability to synthesize antimicrobial compounds. Milk naturally harbors a wide variety of LAB, offering a promising source for identifying strains with biopreservative potential. This study investigated the antagonistic effects, safety characteristics, and technological properties of LAB strains isolated from traditionally fermented milk. Thirty-two dairy samples were analyzed, and the resulting LAB isolates were screened for inhibitory activity against Listeria monocytogenes CECT 4032 and Staphylococcus aureus CECT 976 using agar spot and well diffusion assays. All tested strains exhibited strong antimicrobial effects, with particularly notable inhibition of L. monocytogenes. After phenotypic screening, five representative isolates were selected for molecular identification and further assessment of safety-related attributes, functional capabilities, auto- and co-aggregation properties. 16S rRNA gene sequencing revealed that four strains belonged to the genus Enterococcus, specifically, one E. faecium and three E. durans, while one was classified as a Lactococcus species. Moreover, none of the strains showed proteolytic or lipolytic activities which highlights their potential use in dairy fermentation processes.

1. Introduction

Lactic acid bacteria (LAB) have attracted considerable scientific interest due to their capacity to produce a broad spectrum of antimicrobial substances. These metabolites contribute to the inhibition of undesirable microorganisms, such as spoilage bacteria and foodborne pathogens, thereby enhancing the microbial safety and extending the shelf life of a wide range of food products. Their natural occurrence in fermented foods and recognized safety status further support their application as bio-preservative agents in food systems [1]. These Gram-positive, fermentative bacteria are classified within the phylum Firmicutes, which includes a variety of genera important in food fermentation [2]. The incorporation of LAB into food systems contributes significantly to improving sensory qualities, including flavor, consistency, and nutritional content. Beyond their well-known technological contributions to fermentation, LAB also play a pivotal role in improving food safety. This is largely due to their capacity to inhibit the growth of spoilage microorganisms and pathogenic bacteria through the synthesis of various antimicrobial compounds. By producing such bioactive metabolites, LAB help create an inhospitable environment for undesirable microbes, thereby contributing to the microbiological stability and safety of food products [3,4]. Lactic acid bacteria exhibit antimicrobial activity through the synthesis of various bioactive metabolites. Among the antimicrobial compounds produced by LAB, organic acids, particularly lactic and acetic acid, play a fundamental role in pathogen inhibition. These acids reduce the pH of the surrounding environment, thereby creating conditions that are hostile to the survival and proliferation of many spoilage and pathogenic microorganisms. In addition to organic acids, LAB produce a variety of other antimicrobial substances that contribute to their protective role in food systems. These include diacetyl, which interferes with bacterial metabolism; carbon dioxide (CO₂), which exerts its bacteriostatic action by acidifying the microbial environment and interfering with several essential cellular functions; and hydrogen peroxide, which induces oxidative stress in microbial cells. Moreover, LAB are known for their ability to synthesize bacteriocins, ribosomally produced antimicrobial peptides that exhibit targeted activity against closely related bacteria, including several foodborne pathogens. These compounds often act synergistically, enhancing the overall inhibitory effect against a broad spectrum of spoilage and pathogenic microorganisms. The combined action of these compounds enhances the overall inhibitory potential of LAB in fermented food systems [5]. Among the various antimicrobial substances produced by LAB, bacteriocins are considered especially promising. These peptides exhibit strong inhibitory effects against a wide range of pathogenic bacteria associated with foodborne illnesses. Their potent antimicrobial properties make them promising agents for improving food safety and prolonging product shelf life [6,7]. Lactococcus and Enterococcus species are commonly found in raw and fermented milk, reflecting their strong adaptation to dairy ecosystems [8,9]. Species such as L. garvieae and various subspecies of L. lactis have been reported to produce a broad spectrum of bacteriocins [10]. Notably, L. lactis strains exhibiting antimicrobial activity have gained considerable interest as promising biopreservatives in the dairy industry [11]. Enterococcus species likewise produce enterocins, a group of bacteriocins that exhibit antimicrobial activity against Gram-positive bacteria, including important foodborne pathogens like Listeria spp. [12]. Among Enterococcus species, E. faecalis and E. faecium are the main producers of enterocins. These strains have been extensively studied owning to their reliable ability to generate various types of enterocins with strong antimicrobial effects [13]. Numerous studies have focused on isolating LAB with antimicrobial activity from dairy matrices. For instance, Achemchem et al. [14] reported the isolation of the bacteriocin-producing Enterococcus faecium F58 strain from traditional soft goat cheese, while Ghrairi et al. [15] reported LAB with anti-Listeria activity from Rigouta, a traditional Tunisian cheese. Additionally, Perin, and Nero [16] identified Lactococcus and Enterococcus as predominant genera in the microbial community of raw goat’s milk. They also highlighted the ability of these bacteria to inhibit the growth of Listeria monocytogenes ATCC 7644. Elotmani et al. [17] also reported the presence of LAB with antilisterial activity in Raïb, a traditional Moroccan fermented milk. In light of these findings, the present study aimed to isolate LAB strains from traditionally fermented milk and assess their antimicrobial activity against key foodborne pathogens. Special emphasis was placed on evaluating the ability of these isolates to produce bacteriocins, antimicrobial peptides known for their targeted inhibitory effects against pathogenic and spoilage microorganisms. Additionally, their safety characteristics and technological functionalities were examined to determine their suitability for application in cheese biopreservation.

2. Materials and Methods

2.1. Isolation of LAB from Milk

A total of 32 samples of spontaneously fermented milk were analyzed to isolate LAB. The fermented milk used was from goat (n = 20), cow (n = 8), and camel (n = 4). Fermentation was carried out at 30 °C overnight. Samples were serially diluted in sterile saline to obtain appropriate concentrations for microbial plating and then applied onto two selective culture media, Man, Rogosa, and Sharpe (MRS) agar (Biokar Diagnostics, Allonne, Beauvais Cedex, France) and M17 agar (Biokar Diagnostics) supplemented with 0.5% glucose (GM17). The inoculated plates were incubated at 30 °C for 24 to 48 h. After incubation, colonies showing typical LAB morphology were isolated and purified. The bacterial isolates obtained were subsequently evaluated for their ability to inhibit the growth of specific pathogenic bacteria. All strains were stored at 4 °C for short-term use and as frozen stocks in 35% glycerol at −20 °C for long-term preservation. Before use, the cultures were twice subcultured in their respective broth media at 30 °C.

2.2. Indicator Pathogens and Antimicrobial Spectrum

The inhibitory potential of the isolated strains was assessed using both the agar spot and well diffusion methods, following the methods described by Achemchem et al. [18]. In the agar spot test, 5 µL of overnight LAB cultures grown in either MRS or GM17 broth were carefully spotted onto the surface of solidified agar plates of the corresponding medium. After incubation at 30 °C for 24 h to allow bacterial growth, the plates were overlaid with soft agar previously inoculated with an overnight culture of the indicator organisms. The overlay was poured gently to ensure even distribution across the plate surface. Following a second incubation, inhibition zones around the colonies were observed as indicators of antimicrobial activity.

For the well diffusion assay, LAB strains were cultured overnight at 30 °C in MRS or GM17 broth. The cultures were then centrifuged, and the resulting supernatants were collected to obtain cell-free supernatants (CFS). The CFS pH was adjusted to 6.5 using a 5 M NaOH solution to prevent inhibition due to acidity. Sterile stainless-steel cylinders were used to create wells in Mueller–Hinton Agar (MHA) plates (Biokar Diagnostics). After wells were prepared in the agar medium, each plate was overlaid with soft agar previously inoculated with an overnight culture of the designated indicator microorganism. Once the overlay solidified at room temperature, 100 µL of the prepared CFS was carefully added to each well. To ensure uniform diffusion of antimicrobial compounds into the agar, the plates were first incubated at 4 °C for 2 h, followed by incubation at 37 °C for 16 h to allow the growth of the indicator strain and expression of any inhibitory activity [19]. Antimicrobial effects were assessed by measuring the diameter of the clear inhibition zones around the wells.

2.3. Bacterial Strains Used in This Study

Ten pathogenic bacterial strains were chosen as indicator organisms to assess the antimicrobial activity of the tested isolates (

Table 1. Origin of Indicator Strains.

Strain Code	Species	Origin *
CECT 7467	L. monocytogenes	CECT Collection
CECT 4032	L. monocytogenes	CECT Collection
CECT 5725	L. monocytogenes	CECT Collection
CECT 935	L. monocytogenes	CECT Collection
CECT 976	S. aureus	CECT Collection
CECT 4076	E. coli	CECT Collection
ATCC 25922	E. coli	ATCC Collection
DSMZ 6633	B. subtilis	DSMZ Collection
CECT 704	S. enterica	CECT Collection
CECT 118	P. aeruginosa	CECT Collection
F 58	E. faecium	Our Collection
AE 99	L. lactis	Our Collection

* DSMZ, German Collection of Microorganisms and Cell Cultures; CECT, Spanish Type Culture Collection; ATCC: American Type Culture Collection.

). The selected Gram-positive strains included L. monocytogenes CECT 4032, CECT 7467, CECT 5725, and CECT 935; S. aureus CECT 976; and Bacillus subtilis DSMZ 6633. The Gram-negative group consisted of Pseudomonas aeruginosa CECT 118, Escherichia coli ATCC 25922 and CECT 4076, and Salmonella enterica CECT 704. Several L. monocytogenes strains were used to represent the diversity that may affect their sensitivity to antimicrobial agents. All indicator strains were cultivated in TSB at 37 °C for 18 h before use in the assays. As a positive control, Enterococcus faecium F58, a well-characterized bacteriocin-producing strain, was grown on MRS agar under standard incubation conditions. For proteolytic activity, E. faecium F58 was used as the negative control and L. lactis AE99 as the positive control. L. lactis AE99 was also used as the negative control for lipolytic activity.

2.4. Antimicrobial Screening of LAB

To screen for bacteriocin production, several colonies were randomly picked and transferred onto two sets of agar plates for further analysis [14]. These plates were then incubated anaerobically at 30 °C for 16 to 24 h to allow sufficient bacterial growth. After the initial incubation period, the plates were overlaid with a layer of soft agar (6 mL, 0.75% agar) that had been inoculated with approximately 10⁸ CFU/mL of an overnight culture of the indicator organisms [14,20], either Listeria monocytogenes CECT 4032 or Staphylococcus aureus 976. This concentration was used in the antimicrobial test to evaluate the direct effect of the LAB on the pathogen. These indicator strains were previously cultivated in Tryptic Soy Broth (TSB) (Biokar Diagnostics) under optimal conditions to ensure sufficient growth and viability before use in the overlay assay. Strains that produced clear and measurable inhibition zones were considered to have antimicrobial activity.

2.5. Stability of Bacteriocin-like Activity After Exposure to Proteolytic Enzymes

To evaluate whether the antibacterial compounds produced by the isolated strains were proteinaceous, their sensitivity to proteolytic enzymes was tested. CFS obtained from cultures at the early stationary phase were incubated at 37 °C for 2 h with proteinase K (1 mg/mL), prepared in the buffer recommended by the manufacturer (Qiagen, Hilden, Germany). L. monocytogenes CECT 4032 was used as the indicator strain, while untreated CFS served as negative controls. Residual antibacterial activity in both treated and untreated samples was assessed through the well diffusion assay. Residual activity was assessed based on the presence of clear inhibition zones on the agar.

2.6. Identification of Antagonistic LAB

2.6.1. Phenotypic and Biochemical Identification

Strains producing bacteriocins were first examined using phenotypic methods, including Gram staining, catalase activity testing, and microscopic observation of cellular morphology. Their biochemical characteristics were also evaluated, such as their ability to grow at 10 °C and 45 °C, tolerate 4% and 6.5% NaCl, and grow on Bile Esculin Azide (BEA) agar (Biokar Diagnostics). To achieve more precise taxonomic identification, the isolates were analyzed using the API 20 Strep identification system (bioMérieux, Marcy l’Etoile, France), according to the manufacturer’s guidelines.

2.6.2. Molecular Identification (16S rRNA)

Following the procedure described by Elidrissi et al. [20], genomic DNA was extracted from bacterial cultures and quantified. A fragment of about 1500 base pairs from the 16S rRNA gene was amplified using universal primers in a 25 µL PCR reaction, with standard thermal cycling including an initial denaturation, 35 cycles of denaturation, annealing, extension, and a final elongation step. The PCR products were checked by agarose gel electrophoresis. Purified amplicons were then sequenced using specific primers. The sequencing products were further purified, analyzed by capillary electrophoresis, and the resulting sequences were identified by BLAST comparison against the NCBI database. Phylogenetic analysis was performed in MEGA v11.0 using the Kimura 2-parameter model with 500 bootstrap replicates.

These procedures were performed at the Technical Support Units for Scientific Research (UATRS), affiliated with the National Center for Scientific and Technical Research (CNRST), Morocco.

2.7. Technological Assessment of LAB Strains

2.7.1. Proteolytic and Lipolytic Activities

Proteolytic activity was evaluated by inoculating the isolates onto Trypticase Soy Agar (TSA) (Biokar Diagnostics) containing 1.5% (w/v) skim milk, following the protocol of Achemchem et al. [18]. After incubation period, the presence of clear halos around the colonies was used as an indicator of proteolytic activity.

The lipolytic activity of the isolates was examined by testing them on nutrient agar enriched with 10 g/L Tween 20 and 0.1 g/L calcium chloride, following the protocol established by Albayrak, and Duran [21]. Ten microliters of each culture were placed onto the prepared medium and incubated under anaerobic conditions at 37 °C for 72 h. The detection of lipase production was based on the visual observation of precipitate formation due to the interaction between calcium ions and fatty acids.

2.7.2. Aggregation Abilities

The aggregation properties of the selected LAB strains were evaluated to determine their potential for adhesion and interaction with pathogenic microorganisms, following the procedures outlined by Elidrissi et al. [20] and Zanzan et al. [22].

Auto-aggregation tests were conducted to evaluate the ability of LAB strains to adhere to their own cells. The degree of auto-aggregation was calculated as a percentage using the formula: Auto-aggregation (%): 1 − (A_t/A₀) × 100, where A₀ and A_t represent the initial and final absorbance at 600 nm, respectively.

To determine the co-aggregation capacity between LAB strains and the foodborne pathogen L. monocytogenes CECT 4032, equal volumes of each bacterial culture were combined and incubated at room temperature without agitation for 4 and 24 h. Absorbance measurements were taken for the individual cultures as well as their mixture. Co-aggregation percentage was calculated using: Coaggregation % = ((A_lab + A_pat) − A_mix)/(A_lab + A_pat) × 100, where A_lab + A_pat are the initial absorbances of the LAB and pathogen suspensions, respectively, and A_mix is the absorbance of the mixture at the specified time point.

2.8. Evaluation of Safety-Related Traits

2.8.1. Hemolytic Activity

The bacterial isolates were tested for hemolytic activity by inoculating them onto blood agar containing 5% sheep blood, followed by incubation at 37 °C for 48 h. After incubation, the presence of hemolysis was evaluated [23]. S. aureus CECT 976 served as the positive control.

2.8.2. Antibiotic Susceptibility

The antibiotic susceptibility of the bacterial isolates was evaluated using the disk diffusion method, following the procedure described by Doménech-Sánchez et al. [24]. All isolates were tested against a range of antibiotics.

The antibiotics tested in this research comprised streptomycin (25 µg), vancomycin (30 µg), netilmicin (30 µg), ampicillin (20 µg), ciprofloxacin (5 µg), penicillin G (10 units), erythromycin (15 µg), chloramphenicol (30 µg), fosfomycin (200 µg), fusidic acid (10 µg), tetracycline (30 µg), gentamicin (10 µg), and kanamycin (30 µg). The inhibition zone diameters around the antibiotic disks were measured, and the results were interpreted based on the guidelines provided by CLSI [25] and Mazlumi et al. [26]. Based on the inhibition zone diameters, isolates were classified as susceptible, intermediate, or resistant.

2.8.3. Gelatinase Activity and Biogenic Amine Production

Gelatinase activity was assessed by inoculating 1 µL of an overnight culture onto BHI agar (Biokar Diagnostics) supplemented with 3% (v/v) gelatin, following the protocol described by Amidi-Fazli, and Hanifian [27]. The inoculated plates were incubated at 37 °C for 48 to 72 h, then transferred to 4 °C for 4 h. The appearance of opaque halos surrounding the colonies was interpreted as a positive indication of gelatinase activity.

The ability of the LAB isolates to produce biogenic amines was assessed using a decarboxylase assay. The test was carried out on Maijala agar medium supplemented with 20 g/L histidine, used as the amino acid pre-cursor. After incubation at 30 °C for 48 h, the formation of a purple coloration in and around the bacterial colonies was considered indicative of histamine production [19].

2.9. Statistical Analysis

Statistical analyses were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, with Statistica version 6.0.

3. Results

3.1. Characterization of Lactic Acid Bacteria with Antimicrobial Potential

By analyzing 32 samples, a total of 1536 bacterial strains were isolated. Of these, 148 strains showed antimicrobial activity on solid media, with 82 active against L. monocytogenes and 66 against S. aureus. Among them, 18 LAB strains exhibiting antimicrobial activity in the liquid assay were selected. LAB isolated from dairy-derived samples were subjected to initial screening to assess their antimicrobial potential. This preliminary evaluation employed the double-layer agar technique against two common foodborne pathogens: L. monocytogenes CECT 4032 and S. aureus CECT 976. Isolates that demonstrated inhibitory effects against at least one of these indicator strains were subsequently selected for further analysis. Their capacity to produce antimicrobial metabolites was then investigated using the agar well diffusion method, and the strains that exhibited antimicrobial activity were further tested to determine their antimicrobial spectrum.

Out of all the isolates tested, 18 LAB strains demonstrated significant antimicrobial activity, producing distinct inhibition zones ranging from 11 to 19 mm in diameter against one or both target pathogens. These strains were subjected to basic phenotypic characterization, including Gram staining, catalase testing, and assessment of growth under various environmental conditions (

Table 2. Phenotypic Characteristics of Selected LAB Strains.

Strains	Gram Staining	Catalase Activity	Growth Ability at 10 °C	Growth Ability at 45 °C	Growth Ability in 4% NaCl	Growth Ability in 6.5%NaCl	Growth in Bile-Esculin-Azide Agar
KB1	+	-	+	+	+	+	+
KB2	+	-	+	+	+	+	+
KB3	+	-	+	+	+	+	+
KB4	+	-	-	+	+	+	+
KB5	+	-	+	+	+	+	+
KB6	+	-	+	+	+	+	+
KB7	+	-	+	+	+	+	+
KB8	+	-	+	+	+	+	+
KB9	+	-	+	+	+	+	+
KB10	+	-	+	+	+	+	+
KB11	+	-	+	+	+	+	+
KB12	+	-	+	+	+	+	+
KB13	+	-	+	+	+	+	+
KB14	+	-	+	+	+	+	+
KB15	+	-	+	+	+	+	+
KB16	+	-	+	+	+	+	+
KB17	+	-	+	+	+	+	+
KB18	+	-	+	+	+	+	+

+: Positive reaction or Growth; -: Negative reaction or Lack of growth.

). All selected isolates were Gram-positive, catalase-negative, and exhibited coccoid morphology under microscopic observation, features characteristic of typical LAB.

To further characterize these antimicrobial strains, the API 20 STREP identification system was used. From this group, five representative isolates were selected for molecular identification by 16S rRNA gene sequencing. Sequence alignment revealed a high degree of similarity to known LAB reference strains available in the GenBank database (

Table 3. Genetic Identification of Selected Antibacterial LAB Strains.

Selected Strains	Origin of Isolate	Identification	Number of Accessions	Homology Scores (%)
KB1	Goat’s milk	E. faecium	OR083364	99.41
KB3		E. durans	OR083365	99.93
KB10		E. durans	OR083366	99.10
KB13		E. durans	OR083367	99.20
KB14		L. lactis	OR083368	99.03

). The phylogenetic relationships of these isolates are depicted in Figure 1, highlighting their clustering with closely related species. Based on the 16S rRNA sequence data, the strains were identified as Lactococcus lactis, Enterococcus faecium, and Enterococcus durans.

The tree based on 16S sequences (Figure 1) clearly shows three main groups:

• The Enterococcus KB10, KB13 and KB3 isolates cluster into two well-supported clades (bootstrap values ≥ 97%) with reference strains of E. durans (ED-RBLC2, chromosome BT0139, Mise655, 4599) and E. faecium (strain WS311). This grouping confirms the affiliation of our isolates with the species E. durans and E. faecium and highlights their evolutionary proximity to these reference lineages.
• Isolate KB14 clusters with reference strains of L. lactis (HF677501, Uglich 42) in a distinct clade, separate from Enterococcus, confirming its membership in the genus Lactococcus.
• Escherichia coli U5/41 is used as an outgroup to root the tree and illustrate the divergence between the genera studied.

Thus, the new Figure 1 is no longer limited to a simple presentation of the tree, but explicitly illustrates the clear separation between the genera Enterococcus and Lactococcus and the close relationship of our isolates with the corresponding reference strains, supported by high bootstrap values.

3.2. Antimicrobial Activity

Eighteen strains were assessed for their ability to inhibit different pathogenic microorganisms. Using the spot method, all tested LAB isolates demonstrated clear antimicrobial activity against Listeria spp., indicating a strong antagonistic potential. In addition to this consistent antilisterial effect, several strains exhibited inhibitory activity against a broader panel of pathogens, including S. aureus CECT 976, Bacillus subtilis DSMZ 6633, Escherichia coli ATCC 25922, E. coli CECT 4076, Salmonella enterica CECT 704, and Pseudomonas aeruginosa CECT 118 (

Table A1. Antimicrobial Activity of Lactic Acid Bacteria Isolates Against Standard Strains using the Agar Spot Test.

Strains	KB1	KB2	KB3	KB4	KB5	KB6	KB7	KB8	KB9	KB10	KB11	KB12	KB13	KB14	KB15	KB16	KB17	KB18
L. monocytogenes CECT 7467	30	24	22	22	21	21	23	10	10	10	10	9	9	17	21	20	17	17
L. monocytogenes CECT 4032	14	20	19	19	20	19	20	11	11	11	9	10	10	19	19	18	18	19
L. monocytogenes CECT 5725	22	22	22	21	22	22	21	11	10	11	10	10	9	24	23	25	24	22
L. monocytogenes CECT 935	14	20	19	18	19	19	20	9	10	9	10	9	9	15	15	16	15	15
S. aureus CECT 976	28	18	20	20	19	20	19	13	13	13	13	13	13	17	18	19	18	16
E. coli CECT 4076	14	14	15	14	9	13	11	13	13	16	14	14	13	17	13	15	15	13
E. coli ATCC 25922	13	12	12	12	13	17	17	*	*	*	*	*	*	16	18	18	17	17
B. subtilis DSMZ 6633	16	13	14	13	12	12	12	*	*	*	*	*	*	20	22	22	22	22
S. enterica CECT 704	17	17	18	15	18	15	15	*	*	*	*	*	*	17	20	18	17	16
P. aeruginosa CECT 118	25	13	15	15	9	16	16	*	*	*	*	*	*	20	19	18	18	17

*: unclear halo.

and

Table A2. Inhibition Effect of Selected Lactic Acid bacteria against Foodborne Pathogens using the Agar Well Diffusion Test.

Strains	KB1	KB2	KB3	KB4	KB5	KB6	KB7	KB8	KB9	KB10	KB11	KB12	KB13	KB14	KB15	KB16	KB17	KB18
L. monocytogenes CECT 7467	18.5	14	14.5	14	14	14	14	15	15	13	13	14	14	16	17	16	16	16
L. monocytogenes CECT 4032	19	17	17	17	15	15	15	16.5	15	14.5	14	14.5	14.5	14	15	15	15	14
L. monocytogenes CECT 5725	18	16	16	16	17	17	17	15.5	15.5	14	14	14	14	16	16	17	14	14
L. monocytogenes CECT 935	19	17	17	16.5	17	17	17	16	16.5	14	13.5	14	14	14	14	14	13	14
S. aureus CECT 976	-	-	-	-	-	-	-	-	-	-	-	-	-	12	13	13	12	13
E. coli CECT 4076	-	-	-	-	-	-	-	-	-	-	-	-	-	9	-	-	-	-
E. coli ATCC 25922	-	2	2	2	2	2	2	-	-	-	-	-	-	-	-	-	-	-
B. subtilis DSMZ 6633	-	-	-	-	-	2	2	-	-	-	2	-	2	-	-	-	-	-
S. enterica CECT 704	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
P. aeruginosa CECT 118	18	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

(-) no inhibition.

).

In the agar well diffusion assay, all selected LAB strains exhibited clear inhibitory effects against L. monocytogenes, confirming their ability to produce antimicrobial substances capable of diffusing through the agar medium. No inhibition zones were detected against either of the E. coli strains or S. enterica CECT 704, indicating the absence of measurable activity against these Gram-negative bacteria under the tested conditions.

Strain-specific antimicrobial patterns were observed against other indicator organisms. E. faecium KB1 showed inhibitory activity against P. aeruginosa CECT 118. E. durans strains KB3, KB10, and KB13 inhibited B. subtilis DSMZ 6633, while L. lactis KB14 exhibited activity against S. aureus CECT 976. These results highlight the variability in antimicrobial performance among the tested LAB strains, with some demonstrating broader spectra of activity beyond L. monocytogenes.

The cell-free supernatants from the five selected strains were treated with proteinase K to assess the nature of the antimicrobial compounds. The enzymatic digestion neutralized the inhibitory activity, indicating that the active substances were proteinaceous, likely corresponding to bacteriocins (Figure 2).

3.3. Safety and Functional Characteristics

None of the five tested strains exhibited gelatinase or hemolytic activity, indicating the absence of these potential virulence factors.

Table 4. Antibiotic Susceptibility of Selected LAB Strains.

Antibiotic	KB1	KB3	KB10	KB13	KB14
Vancomycin	I	S	S	I	S
Fosfomycin	S	S	S	S	S
Penicillin G	R	S	S	S	S
Ampicillin	I	S	S	S	S
Ciprofloxacin	I	S	I	I	I
Fusidic Acid	I	S	S	S	I
Streptomycin	R	R	I	I	I
Gentamicin	R	S	I	I	I
Chloramphenicol	S	S	S	R	S
Netilmicin	R	S	I	I	I
Erythromycin	R	S	S	S	S
Tetracycline	S	S	S	S	S
Kanamycin	R	S	I	I	I

R: resistant (zone diameter < 12.4 mm); I: intermediate susceptibility (zone diameter 12.5–17.4 mm); S: susceptible (zone diameter > 17.5 mm) [26].

summarizes the antibiotic susceptibility results for the five isolates evaluated in this research. All strains were susceptible to fosfomycin and tetracycline. However, strain KB1 exhibited resistance to penicillin G, streptomycin, gentamicin, netilmicin, and kanamycin. Strain KB3 showed resistance only to streptomycin, while KB13 was the only isolate resistant to chloramphenicol. In contrast, strains KB14 and KB10 exhibited favorable susceptibility profiles, being either sensitive or intermediately susceptible to all 13 antibiotics tested. Additionally, none of the isolates showed proteolytic or lipolytic activity, further supporting their safety potential for use in food-related applications.

3.4. Aggregation Capacity of LAB Isolates

The ability of the five selected LAB isolates to auto-aggregate was evaluated over time. After 4 h of incubation, auto-aggregation levels varied considerably among the strains, ranging from 0.35 ± 0.50% to 27.53 ± 6.46% (Figure 3). The E. durans KB13 strain exhibited a significant difference relative to the other strains after 4 h of incubation (p < 0.05). A substantial increase was observed after 24 h, with values reaching between 28.21 ± 7.25% and 59.56 ± 1.48%, indicating a time-dependent enhancement in cell-to-cell aggregation capacity. This characteristic is often associated with improved colonization potential and stability in microbial communities.

Co-aggregation with L. monocytogenes CECT 4032 also revealed strain-specific differences (Figure 4). Among the tested isolates, L. lactis KB14 showed the lowest level of co-aggregation, while E. durans KB3 exhibited the highest interaction with the pathogen. After 24 h, co-aggregation values ranged from 30.00 ± 0.38% to 55.52 ± 5.96%, with statistically significant differences between strains. The Lactococcus strain showed a significant difference compared to the Enterococcus strains after 24 h (p < 0.05). These findings suggest varying potential among isolates for pathogen exclusion through co-aggregation, which may contribute to their probiotic or bioprotective roles.

4. Discussion

4.1. Antimicrobial Potential and Safety Assessment of LAB Strains

Bacteriocinogenic LAB were isolated from a variety of fermented milk samples collected Morocco. A total of eighteen bacteriocinogenic strains were successfully obtained from fermented goat’s milk. These strains were screened for antimicrobial activity against indicator pathogens using well diffusion assays. All eighteen strains demonstrated clear inhibition zones, indicating their ability to produce antimicrobial compounds, likely bacteriocins, capable of inhibiting the growth of susceptible microorganisms.

In the present study, LAB isolated from raw goat’s milk exhibited pronounced antimicrobial activity against L. monocytogenes, corroborating previous findings that emphasize the inhibitory potential of dairy-derived LAB [16,28,29,30,31]. Prior research has particularly highlighted the antimicrobial capabilities of E. faecium, E. durans, and L. lactis, especially those isolated from traditional artisanal cheeses. These strains have demonstrated the ability to inhibit not only L. monocytogenes, but also a range of other foodborne pathogens, including Bacillus cereus and P. aeruginosa [14,32,33,34,35,36,37].

Antibiotic susceptibility testing revealed that none of the isolates exhibited resistance to vancomycin, in agreement with previously published data [21,37,38]. Notably, L. lactis KB14, isolated in this study, was susceptible to ampicillin, chloramphenicol, and erythromycin, in line with the findings reported by Kazancıgil et al. [39].

Collectively, these findings support the potential application of the studied LAB strains as natural biopreservatives in cheese production. Their strong antimicrobial activity and favorable antibiotic resistance profiles suggest that they could contribute to enhancing food safety and extending shelf life in fermented dairy products.

4.2. Proteolytic and Lipolytic Activities

In the context of proteolytic and lipolytic activities, our results are consistent with those of Albayrak, and Duran [21], who reported that Enterococcus strains isolated from dairy sources lacked proteolytic activity. This absence of proteolytic function suggests that certain Enterococcus spp. may have limited capacity to degrade milk proteins, potentially preserving the structural integrity and sensory attributes of fermented dairy products. However, proteolytic activity is not universally absent in this genus. Several other studies have reported the presence of proteolytic enzymes in Enterococcus species, highlighting strain-dependent variability. For instance, Achemchem et al. [18], de Sousa et al. [35], Mercha et al. [40], and Islam et al. [41] observed proteolytic activity in different Enterococcus strains, suggesting that some isolates may contribute to flavor development or textural changes.

Regarding Lactococcus strains, similar variability in proteolytic capabilities has been noted. With regard to Lactococcus strains, Cheng et al. [42], reported that the isolates tested exhibited proteolytic activity. Conversely, Allam et al. [43] identified two Lactococcus strains that lacked this enzymatic function, reinforcing the notion that proteolytic capacity is strain-specific and must be evaluated individually when selecting candidates for starter or adjunct cultures.

The absence of proteolytic activity in the strains may be related to several factors, including the absence or variability of certain genes of the proteolytic system (PrtP, peptidases, transporters), which can vary among LAB strains [44]. Furthermore, the activity of intra- and extracellular proteolytic enzymes strongly depends on environmental conditions [45]. This absence may be due to genetic factors, environmental factors, and the influence of the method used.

In our study, none of the tested strains displayed lipolytic activity, as no visible hydrolysis of lipids was observed. This finding aligns with previous reports indicating that nisin-producing L. lactis strains generally lack lipolytic enzymes [11]. Similarly, multifunctional LAB strains isolated from artisanal cheeses have also been shown to be non-lipolytic [21]. The absence of lipolytic activity is particularly advantageous in dairy fermentation, as excessive lipid hydrolysis can lead to off-flavors or rancid notes, compromising product quality.

The absence of proteolytic and lipolytic activities in the selected strains presents both advantages and limitations. On the one hand, the lack of these enzymatic activities is desirable from a safety and technological perspective, as it reduces the risk of excessive protein or fat degradation that could lead to textural defects or the formation of undesirable metabolites. On the other hand, this characteristic may also limit the strains’ contribution to flavor development during cheese ripening. Indeed, LAB contribute to flavor development in fermented foods primarily through their proteolytic and lipolytic activities, which generate key aroma-forming compounds [46,47,48].

4.3. Auto-Aggregation and Co-Aggregation Capabilities of LAB Strains

The capacity of LAB strains to auto-aggregate and co-aggregate is widely recognized as a key functional characteristic that underpins their ability to persist and colonize host environments, such as the gastrointestinal tract [49]. These aggregation properties contribute not only to microbial adhesion and biofilm formation but also to the exclusion of potential pathogens through competitive mechanisms [50]. In the present study, the evaluated LAB strains exhibited varying levels of auto-aggregation after 4 h of incubation, with some isolates demonstrating significantly stronger aggregation capabilities than others. These results align with those reported in the literature [39,51,52]. A longer incubation period (24 h) led to a noticeable enhancement in auto-aggregation capacity for all tested strains, particularly for E. durans KB3, which exhibited the highest level of auto-aggregation. This increase over time may reflect enhanced expression of surface proteins or exopolysaccharides that promote cell–cell adhesion during prolonged growth phases.

In terms of co-aggregation, the tested strains also displayed a time-dependent increase in their ability to aggregate with L. monocytogenes CECT 4032, in agreement with patterns previously observed in the literature [53,54]. Notably, the co-aggregation ability of the Lactococcus strain was significantly lower than that of Enterococcus strains after 24 h of incubation, suggesting species-specific differences in surface structures or in the composition of extracellular polymeric substances involved in cell interactions.

Both auto- and co-aggregation are considered beneficial traits for probiotic and bioprotective functions. Auto-aggregation is often associated with enhanced biofilm-forming ability, which facilitates stronger adhesion to intestinal epithelial surfaces and promotes colonization. Co-aggregation, on the other hand, plays a crucial role in the exclusion of enteric pathogens by forming physical barriers or interfering with pathogen adhesion, thus contributing to microbial equilibrium and host protection [31,55]. Collectively, these characteristics underline the promising potential of the selected LAB strains not only as probiotics capable of colonizing and persisting in the gastrointestinal environment but also as effective functional cultures for application in food systems, particularly for improving microbial safety and shelf life.

4.4. Protein-Based Features of Antimicrobial Substances from LAB

The LAB strains evaluated exhibited antimicrobial activity suggestive of bacteriocin production. These inhibitory effects were eliminated following treatment with a proteolytic enzyme (Figure 2), confirming the proteinaceous nature of the active compounds. Bacteriocin-producing LAB are increasingly recognized as promising natural biopreservatives in the food industry, particularly in the dairy sector, owing to their ability to enhance microbial safety and extend shelf life without compromising product quality.

5. Conclusions

In summary, these findings represent an important step toward identifying effective LAB strains for biopreservation. To fully harness their application potential, future research should focus on the purification and comprehensive biochemical characterization of the bacteriocins and other antimicrobial metabolites produced by these strains. Understanding their molecular structure, mechanisms of action, and stability under various food processing and storage conditions will be essential for their effective integration into industrial processes.

Moreover, it is crucial to evaluate the effectiveness of these strains in real food matrices under realistic storage and production conditions. Such trials should assess not only microbial inhibition, but also the impact of LAB on sensory attributes (e.g., flavor, texture, aroma) and product shelf life. Investigating their interactions with the native microbiota and technological parameters will be vital for successful incorporation into commercial cheese production.

In conclusion, the selected LAB strains demonstrate both safety and functionality, supporting their application as natural preservatives in the dairy industry. Their future integration into sustainable food preservation strategies may contribute to cleaner-label products, enhanced food safety, and extended shelf life of both artisanal and industrial fermented cheeses.