Probiotic Potential and Characterization of Enterococcus faecium Strains Isolated from Camel Milk: Implications for Animal Health and Dairy Products

Abstract

In this study, 62 lactic acid bacteria (LAB) strains were isolated from raw camel milk and evaluated for their probiotic potential. The strains exhibited significant variability in their ability to withstand simulated gastrointestinal conditions. Of the isolates, only 26 survived exposure to pH 2, and just 10 were tolerant to 0.3% bile salts. Partial sequencing of the 16S rRNA gene identified all the strains as belonging to the species Enterococcus faecium. Several probiotic traits were assessed, including adhesion to gastric mucin and STC-1 intestinal epithelial cells, as well as auto-aggregation and co-aggregation capacities. Although adhesion to hydrophobic solvents such as chloroform and ethyl acetate was generally low to moderate, all the strains demonstrated strong adhesion to gastric mucin, exceeding 60% at all the growth stages. Notably, two strains—SCC1-33 and SLch6—showed particularly high adhesion to STC-1 cells, with values of 7.8 × 10³ and 4.2 × 10³ CFU/mL, respectively. The strains also exhibited promising aggregation properties, with auto-aggregation and co-aggregation ranging between 33.10% and 63.10%. Furthermore, all the isolates displayed antagonistic activity against Listeria innocua, Micrococcus luteus, and Escherichia coli. Cytotoxicity assays confirmed that none of the tested strains had harmful effects on STC-1 cells, indicating their safety and supporting their potential application as probiotics.

1. Introduction

There is a growing demand for safe foods that contribute to well-being and promote longevity. Among these, dairy products containing probiotic bacteria are highly sought after due to the health benefits associated with the consumption of specific, well-characterized probiotic strains [1]. Several strains of lactic acid bacteria (LAB) are commonly used as probiotics, with the most extensively studied LAB strains belonging to the genera Lactobacillus, Streptococcus, Lactococcus, and Enterococcus. In addition, Bifidobacterium species—although not classified as LAB—are well recognized for their health-promoting properties and are considered valuable probiotics [2].

Studies on the beneficial properties of probiotics have demonstrated that certain strains can serve as alternatives to antibiotic therapy. For example, Lactobacillus rhamnosus GG has been shown to reduce the duration of acute gastroenteritis in children [3], while Saccharomyces boulardii is commonly used to prevent antibiotic-associated diarrhea and recurrent Clostridioides difficile infections [4]. These probiotics can be administered to prevent infectious diseases, enhance the barrier function of the gut microflora, and modulate the innate immune response by promoting non-specific defense mechanisms, a phenomenon sometimes referred to as trained immunity [5]. This is particularly important, as antibiotic treatments can disrupt the balance of the resident gut flora and act as immunosuppressors [6]. To achieve the desired health benefits, probiotics must successfully colonize, adhere to, and remain viable and active in different parts of the intestines [7]. These strains must survive the harsh conditions of the stomach and small intestine in order to reach the colonization site. In particular, they must overcome biological barriers such as stomach acid and bile in the intestines [8].

Bioprospecting for efficient probiotic strains from underexplored ecological niches is a promising research avenue due to the high diversity among probiotic microorganisms [9]. Camel milk represents one such niche, offering potential as a source of lactic acid bacteria that are not yet fully characterized. Like other types of milk, camel milk contains LAB such as Lactobacillus, Lactococcus, and Enterococcus species [10]. However, its unique biochemical composition compared to cow milk [11], as well as its origin in arid, low-human-impact environments, suggests it may harbor a distinct microbiota with unique probiotic properties. Camel milk has been associated with therapeutic benefits in conditions such as food allergies and diabetes [12]; moreover, camel milk contains natural antimicrobial compounds such as lactoferrin, lysozyme, immunoglobulins, and bioactive peptides, which contribute to its ability to inhibit pathogenic bacteria [13]. Notably, Al-Otaibi et al. [14] demonstrated that LAB strains isolated from camel milk can inhibit Escherichia coli and reduce the adhesion of Staphylococcus aureus in the gastrointestinal tract [15]. However, research on the probiotic properties of LAB strains isolated from camel milk remains limited. Therefore, the aim of this study was to isolate and characterize LAB strains from camel milk and to evaluate their probiotic potential based on in vitro functional and safety criteria.

2. Materials and Methods

2.1. Isolation and Culture of Bacterial Strains

Lactic acid bacteria (LAB) strains were isolated from raw camel milk (Camelus dromedarius) collected from a healthy camel herd at the Arid Lands Institute (IRA) in Medenine, Tunisia. Approximately 50 mL of milk was aseptically collected in sterile containers and immediately stored in insulated boxes with ice packs (4 °C) to maintain the cold chain during transport to the laboratory. The samples were processed within 4 h of collection. LAB were cultured on de Man Rogosa and Sharpe (MRS) agar (Pronadisa, Madrid, Spain) and incubated at 37 °C for 24 to 48 h under aerobic conditions. Colonies with distinct morphologies were selected and subjected to conventional phenotypic identification tests, including Gram staining, catalase activity, and motility assays, following standard microbiological protocols [16].

Gram-positive, catalase-negative, and non-motile isolates were selected and stored in MRS broth supplemented with 30% sterile glycerol as a cryoprotectant at −80 °C. Prior to analysis, the purified cultures were revived by sub-culturing twice in MRS broth.

2.2. Molecular Identification Using 16S rRNA Gene

Genomic DNA was extracted from overnight LAB cultures using the GeneJET Genomic DNA Purification Kit (Fermentas, Cambridge, UK), following the manufacturer’s protocol. The concentration and purity of extracted DNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).

PCR amplification of the 16S rRNA gene was performed in a 50 μL reaction volume containing 0.5 μL of template DNA, 2.5 μL each of forward (S1: 5′-AGAGTTTGATC(A,C)TGGCTCAG-3′) and reverse (S2: 5′-GG(A,C)TACCTTGTTACGA(T,C)TTC-3′) primers (10 μM), 2 μL of dNTP mix (25 mM), 4 μL of MgCl₂ (25 mM), 5 μL of 10× PCR buffer, and 1 μL of Taq DNA polymerase (5 U/μL; Thermo Scientific), with nuclease-free water added to a final volume of 50 μL. PCR reactions were conducted in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA).

The PCR amplification was carried out using the following thermal cycling program: an initial denaturation step at 94 °C for 5 min; followed by 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min; with a final extension at 72 °C for 10 min. The PCR products were verified by electrophoresis on a 1.5% agarose gel stained with ethidium bromide and visualized under UV light using a GelDoc™ EZ system (Bio-Rad, Hercules, CA, USA) [17,18]. The expected amplicon size of approximately 1500 bp was confirmed before proceeding with downstream analyses. The resulting amplicons were then cloned into the pGEM-T Easy Vector System (Promega Corp., Charbonnières-les-Bains, France), followed by plasmid extraction using the GeneJET Plasmid Mini Prep (Thermo Scientific, Surrey, UK). Sequencing of the amplicons was performed by the sequencing facility at Eurofins (Ebersberg, Germany).

The obtained nucleotide sequences were analyzed using the BioEdit software version 7.2.5 and compared against sequences in the NCBI database using the BLAST tool software version 2.17.0 (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine identity percentages.

2.3. Assessment of Probiotic Potential

2.3.1. Tolerance to pH and Bile Salts

The isolated LAB strains were evaluated for their ability to withstand low pH and bile salts. A pH of 2, representative of gastric conditions, was used in the experiment. After 16 to 18 h of culture under aerobic conditions, the cells were harvested by centrifugation at 5000× g for 10 min at 4 °C. The resulting pellets were washed once in phosphate-buffered saline (PBS), then resuspended in PBS at pH 2, and incubated at 37 °C. After 4 h of incubation, colonies were counted, and the number of LAB was calculated following the standard ISO 15214 ([19]). The survival rate of LAB strains was determined by comparing the number of colonies grown on MRS agar after 4 h of incubation in PBS at pH 2 to the number of colonies obtained from a control incubation in PBS at physiological pH (7.0). This approach allowed the assessment of LAB viability under acidic conditions relative to neutral pH.

Strains that showed resistance to low pH were subsequently tested for bile tolerance. Considering that the average intestinal bile concentration is approximately 0.3% (w/v) and food typically stays in the small intestine for about 4 h [20], the experiment was conducted at this concentration of bile for 4 h. MRS medium containing 0.3% bile (Oxoid, Cheshire, UK) was inoculated with active cultures (incubated for 16–18 h). During the 4 h incubation, viable cells were enumerated every hour using the pour plate technique, and growth was monitored by measuring the optical density (OD600). Lactococcus lactis, a non-probiotic strain, was used as a negative control due to its well-documented sensitivity to bile salts and poor survival under gastrointestinal conditions, as reported in previous studies [21,22].

2.3.2. Assessment of Adhesion Properties

a. 

Adhesion to Hydrocarbons

The bacterial adhesion to hydrocarbons test (BATH) was conducted following the methods of Collado et al. [23] and Kos et al. [24]. The cells were washed once with phosphate-buffered saline (PBS, pH 7.2) and resuspended in the same buffer to an optical density (OD600) of approximately 0.25 ± 0.05 (OD₀) to standardize the bacterial concentration (10⁷–10⁸ CFU/mL). An equal volume of solvent was then added. The two-phase system was mixed vigorously for 5 min. After 1 h of incubation at room temperature, the aqueous phase was separated, and its optical density at 600 nm (OD₁) was measured. The percentage of bacterial adhesion to the solvent was calculated using the following formula:

Adhesion % = ( (OD₀ − OD₁) OD₀) × 100

Two solvents were tested in this study: chloroform, a monopolar and acidic solvent, and ethyl acetate, a monopolar and basic solvent.

b. 

Bacterial Adhesion to Gastric Mucin

The strains were evaluated for their ability to adhere to immobilized porcine gastric mucin (Type III, Sigma-Aldrich, St. Louis, MO, USA) in 96-well polystyrene microplates (Nunc Maxisorp, Roskilde, Denmark). Each well was coated with 100 µL of porcine gastric mucin solution (10 mg/mL) in sterile PBS (pH 7) and incubated overnight at 4 °C. The wells were then washed twice with 200 µL of sterile PBS to remove any unbound mucin. To block non-specific binding sites, 200 µL of bovine serum albumin solution (2% w/v in PBS) was added, and the microplates were incubated for 2 h at 37 °C. Following this, the wells were washed twice with 200 µL of sterile PBS.

The bacterial cells were harvested at three different growth stages (9, 12, and 24 h) by centrifugation at 3500× g for 5 min at 4 °C. The pellets were washed twice with 1 mL of 0.1 M Tris-HCl (pH 7.5) and then centrifuged again. The cells were resuspended in PBS and diluted to achieve an optical density (OD600) of 0.1 ± 0.02, corresponding to approximately 3 × 10⁷ CFU/mL. A 100 µL aliquot of each bacterial suspension was added to the wells, and the microplates were incubated for 1 h at 37 °C. After incubation, the liquid was removed by pipetting, and each well was washed five times with 200 µL of PBS.

To desorb the adhered cells, 200 µL of 0.5% Triton X-100 solution (v/v) was added for 20 min at room temperature, with orbital stirring at 150 rpm. The number of desorbed bacteria was determined by plating the solution onto MRS agar plates. All the adhesion tests were performed in triplicate.

c. 

Adhesion to Intestinal Cell Line (STC-1)

Adhesion to the mouse intestinal endocrine tumor cell line (STC-1) was assessed. The STC-1 cells were cultured in DMEM medium (Dulbecco’s Modified Eagle’s Minimal Essential Medium), supplemented with 10% (v/v) calf serum, 100 µg/mL streptomycin, and 100 IU/mL penicillin (Gibco, Waltham, MA, USA), and maintained at 37 °C in a 95% air/5% CO₂ atmosphere. The medium was replaced every 2 days, and the cells were used after reaching full confluence on day 15.

For the adhesion assay, the cells were seeded at a density of 5 × 10⁴ cells per well in 24-well plates and incubated at 37 °C until a confluent monolayer was formed. Prior to bacterial inoculation, the wells were washed with pre-warmed medium to remove any antibiotics. Bacteria were grown in MRS medium for 18 h at 37 °C and then washed twice in 100 mM phosphate buffer (pH 7.0). The bacterial suspension was diluted in DMEM to obtain a final concentration of 1 × 10⁷ CFU/mL. One mL of the bacterial suspension was added to each well.

After a 2 h incubation, free bacteria were removed by washing with 1 mL of pre-warmed phosphate buffer. To detach the adherent bacteria, 1 mL of 1% Triton X-100 was added, and after 10 min of incubation, serial dilutions of the solution were plated onto MRS agar. Plates were incubated for 48 h at 37 °C. This test was performed in triplicate.

2.3.3. Auto-Aggregation Test

Auto-aggregation assays were performed following the method described by Del Re et al. [25], with some modifications. LAB strains were cultured in MRS broth for 18 h at 37 °C. The cells were harvested by centrifugation at 5000× g for 15 min, washed twice, and then resuspended in phosphate-buffered saline (PBS). The suspension was adjusted to approximately 10⁸ CFU/mL using optical density measurements at 600 nm (OD₆₀₀), previously calibrated against standard plate counts. Cell suspensions (4 mL) were then mixed by vortexing for 10 s. After an incubation period of 4 h at 37 °C, 0.1 mL of the upper suspension was transferred to a new tube containing 3.9 mL of PBS. The optical density (OD) was measured at 600 nm. The auto-aggregation rate was calculated using the following formula:

Aggregation % = (1 − OD_{upper suspension}/OD_{total bacterial suspension}) × 100

2.3.4. Co-Aggregation with Saccharomyces Cerevisiae

Co-aggregation tests were performed following the method described by Collado et al. [23]. Briefly, bacterial suspensions were prepared as described for the auto-aggregation assays. Equal volumes (100 µL each) of bacterial suspensions from different Enterococcus strains and Saccharomyces cerevisiae (grown in Sabouraud Dextrose Broth, Sigma-Aldrich, St. Louis, MO, USA, for 24 h) were mixed and incubated at 20 °C and 37 °C without agitation. Pure bacterial suspensions (200 µL each) were incubated under similar conditions to serve as controls for self-flocculation.

After 4 h of incubation, the optical density (OD) of the mixtures and the pure bacterial suspensions was measured at 600 nm. The co-aggregation percentage was calculated using the following formula described by De Gregorio et al. [26]:

Co-aggregation % = (OD_Sac + OD_Bact) − OD_Mix(OD_Sac + OD_Bact) × 100

where

OD_Sac represents the OD₆₀₀ of S. cerevisiae at time T₀.

OD_Bact represents the OD₆₀₀ of the bacterial suspension at time T₀.

OD_Mix represents the OD₆₀₀ of the mixture after 4 h of incubation.

2.3.5. Survival Under Simulated In Vitro Digestion Conditions

The survival of lactic acid bacteria (LAB) under simulated gastrointestinal conditions was evaluated using a modified version of the method described by Seiquer et al. [27]. Each LAB strain was first cultured in sterile skimmed milk at 30 °C for 8 h to mimic a dairy-based carrier matrix. After fermentation, 1 g of the cultured milk was diluted 1:10 (w/v) in sterile phosphate-buffered saline (PBS, pH 7.2). To simulate gastric digestion, the pH was adjusted to 3.0 using 1 N HCl, and porcine pepsin (Sigma-Aldrich, ≥250 units/mg protein) was added to a final concentration of 5% (w/v). The samples were incubated at 37 °C with constant agitation at 110 rpm for 90 min.

For the intestinal phase, the pH was increased to 6.0 using 1 N NaOH. Pancreatin (Sigma-Aldrich, containing trypsin ≥100 USP units/mg) and bile salts (Oxoid) were added to final concentrations of 0.1% and 0.3% (w/v), respectively. The mixtures were incubated at 37 °C for 150 min under agitation (110 rpm). Aliquots were collected at the start and end of each digestion phase. Viable cell counts were determined by serial dilution and plating on MRS agar, followed by anaerobic incubation at 37 °C for 48 h. All assays were performed in triplicate.

To account for differences in initial bacterial concentrations among isolates, the survival rate was expressed as the log reduction and log survival percentage, calculated as follows:

Log Survival (%) = (log10CFU/mL before digestion/log10CFU/mL after digestion) × 100

This normalization allowed for the consistent comparison of digestion resistance among strains, regardless of initial viable counts.

2.3.6. Antagonistic Activity

The antibacterial activity of the selected isolates was evaluated using the agar spot-on-lawn test, as described by Schillinger and Lücke [28] with some modifications. The indicator bacteria used in this study included Listeria innocua (ATCC 33090), Micrococcus luteus (ATCC 4698), and Escherichia coli (ATCC 25922). One microliter of each overnight culture of the selected lactic acid bacteria (LAB) was spotted onto MRS plates containing 0.2% glucose and 1.2% agar. These plates were then incubated under anaerobic conditions for 48 h to allow colony development. A 0.25 mL portion of a 1:10 dilution of an overnight culture of the indicator bacteria was added to 9 mL of Brain Heart Infusion (Merck, Darmstadt, Germany) soft agar (0.7% agar). The medium was then immediately poured over the MRS plate on which the tested LAB strain had grown. The plates were incubated anaerobically at 37 °C for 24 h. Antibacterial activity was determined based on the clear inhibition zone around the LAB colonies, which was calculated by measuring the difference between the total inhibition zone diameter and the diameter of the growth spot of the selected strains. Zones with diameters larger than 1 mm were considered to exhibit antagonistic activity, as per the criteria outlined by Yavuzdurmaz [29]. To further assess the antagonistic activity, 1 µL of the LAB culture was replaced with 1 µL of nisin solution.

2.3.7. Safety Assessment of Enterococcus faecium Strains

a. 

Antibiotic Susceptibility

The antibiotic susceptibility of the Enterococcus faecium strains was determined using the agar disk diffusion method. The strains were grown overnight in MRS broth at 37 °C, and 100 µL of the diluted culture (approximately 10⁶ viable cells) was streaked onto MRS agar plates. The following antibiotics were used at the indicated concentrations: 30 µg tetracycline, 10 µg ampicillin, 1000 µg kanamycin, 15 µg erythromycin, 30 µg rifampicin, and 30 µg vancomycin. The plates were incubated at 37 °C under anaerobic conditions for 18 h, and the inhibition zones were measured. According to CLSI zone diameter interpretative standards, strains were considered resistant if the inhibition zone diameter was less than 17 mm for vancomycin, erythromycin, and tetracycline; less than 16 mm for ampicillin; and less than 10 mm for kanamycin [30].

b. 

Cytotoxic Assay on STC-1 Cells

The cytotoxicity of the Enterococcus faecium strains was evaluated on the STC-1 cells according to the method of Rindi et al. [31]. The cells were seeded in a 96-well plate at 7000 cells per well in 150 µL of DMEM medium (Dulbecco’s Modified Eagle Medium containing 4.5 g/L glucose, 5% fetal bovine serum, 2 mM glutamine, 100 µg/mL streptomycin, and 100 IU/mL penicillin) and cultured for approximately 72 h at 37 °C in a 95% air/5% CO₂ atmosphere until they reached confluence. The DMEM medium was then removed, and the cells were washed twice with minimum medium (without serum or antibiotics). A volume of 80 µL of minimum medium containing bacterial strains at concentrations of 10⁵ or 10⁷ CFU/mL was added, with sterile minimum medium serving as the control. Twenty µL of propidium iodide at a concentration of 5 µg/mL was added to each well. Fluorescence emission was monitored over 60 min, every 30 s, using excitation/emission wavelengths of 575/615 nm with 5 nm slits, on a spectrofluorimeter (SAFAS, Monaco, Monaco). The results were expressed as the fluorescence emission “fold of control” compared to non-treated cells.

2.4. Statistical Analysis

All the experiments were performed in triplicate, and the results are presented as mean ± standard deviation. Statistical analyses were conducted using one-way analysis of variance (ANOVA) to determine significant differences among strains for each test. When ANOVA indicated significant differences (p < 0.05), Tukey’s Honestly Significant Difference (HSD) post hoc test was applied to identify specific group differences. Different letters (a, b, c) were used to denote statistically distinct groups.

3. Results and Discussion

The applied experimental plan focused on assessing the in vitro probiotic potential of LAB isolates through a series of functional assays. These included tests for acid and bile salt tolerance, antimicrobial activity, antibiotic susceptibility, auto-aggregation, and hydrophobicity. Together, these assays aimed to evaluate the strains’ ability to survive gastrointestinal conditions and exhibit beneficial probiotic properties.

3.1. Recovery and Preliminary Identification of Lactic Acid Bacteria

A total of 62 strains were isolated from camel milk using MRS agar incubated at 37 °C under anaerobic conditions. All the isolates were Gram-positive, non-motile cocci, and catalase-negative, which are typical preliminary characteristics of lactic acid bacteria.

3.2. Functional Screening: Acid and Bile Salt Resistance

Resistance to low pH is a key criterion in selecting potential probiotic strains [32], as they must survive the harsh conditions of the stomach to reach the small intestine [33]. In this study, only 26 isolates demonstrated tolerance to acidic conditions and were subsequently tested for bile salt resistance.

At this stage, ten strains were selected for further probiotic evaluation and molecular identification. These strains exhibited good tolerance to bile salts: eight strains (SSC1-2, SCC1-6, SCC1-8, SCC1-13, SCC1-15, SCC1-24, SCC1-33, and SLch14) maintained survival rates above 0.5 OD600 units after incubation, while two strains (SCC1-7 and SLch6) showed survival rates exceeding 1 OD600 unit.

3.3. Genetic Characterization of Isolates by 16S rRNA Sequencing

A ~1500 bp fragment of the 5′ region of the 16S rRNA gene was amplified and sequenced for the ten selected isolates. A sequence analysis using the NCBI database revealed that all ten isolates shared 99% identity with Enterococcus faecium (

Table 1. Identification of LAB isolates from camel milk and their closest BLASTn matches.

Isolate Code	Closest Species Match	% of Similarity	Closest GenBank Accession Number
SCC1-2	E. faecium	99%	JN560903.1
SCC1-6	E. faecium	99%	KF149320.1
SCC1-7	E. faecium	99%	JX847611.1
SCC1-8	E. faecium	99%	JQ726533.1
SCC1-13	E. faecium	99%	EU878170.1
SCC1-15	E. faecium	99%	KC422716.1
SCC1-24	E. faecium	99%	JN560911.1
SCC1-33	E. faecium	99%	JN560898.1
SLch6	E. faecium	99%	HM162421.1
SLch14	E. faecium	99%	AY587799.1

Accession numbers correspond to the closest matches identified via BLASTn analysis.

). These results align with previous studies reporting that lactic acid bacteria isolated from camel milk are predominantly Enterococcus species [34]. Specifically, E. faecium has been previously isolated from Egyptian camel milk [35], as well as from both raw and fermented Bactrian camel milk [36].

3.4. Evaluation of Adhesion to Organic Solvents, Gastric Mucin, and Intestinal Cells

3.4.1. Bacterial Adhesion to Hydrocarbons (BATH) Assay of E. faecium Strains

Bacterial adhesion to organic solvents is involved in various interfacial phenomena, including microbial attachment to host tissues and surfaces. In this study, the adhesion of Enterococcus faecium strains to chloroform and ethyl acetate was assessed to determine the Lewis acid-base characteristics of their cell surfaces. Chloroform, an acidic solvent and electron acceptor, and ethyl acetate, a basic solvent and electron donor, were used following the methodology described by Kos et al. [24].

Our findings showed that the tested strains exhibited low to moderate adhesion to both solvents, with adhesion percentages generally below 40% (

Table 2. Adhesion to organic solvents (%), co-aggregation (%) with S. cerevisae at 20 °C and 37 °C, and auto-aggregation of the different Ent. faecium strains.

Strains	Adhesion to Ethyl Acetate	Adhesion to Chloroform	Co-Aggregation with S. cerevisae		Auto-Aggregation
			20 °C	37 °C
SCC1-2	16 b ± 5.66	12 b ± 5.66	61.77 a ± 11.05	46.78 a ± 2.16	41.03 b ± 11.1
SCC1-6	26.0 ab ± 2.83	16.7 ab ± 2.62	58.10 ab ± 13.38	45.07 a ± 2.23	62.76 a± 3.0
SCC1-7	20.7 ab ± 9.75	14.4 ab ± 4.08	48.74 b ± 1.99	43.31 ab ± 12.29	59.66 a± 6.3
SCC1-8	31.8 a ± 10.71	18.8 ab ± 2.95	48.43 b ± 6.93	44.94 ab ± 18.76	41.7 b ± 9.6
SCC1-13	00 c ± 00	30.4 a ± 7.58	62.59 a ± 0.68	43.41 ab ± 0.1	49.1 a ± 10.0
SCC1-15	25.0 ab ± 8.16	28.6 a ± 5.05	55.28 ab ± 4.13	43.98 ab ± 0.29	43.0 b ± 5.0
SCC1-24	34.5 a ± 4.88	14.8 ab ± 5.24	44.40 b ± 7.2	43.46 ab ± 2.08	59.3 a ± 3.4
SCC1-33	18 b ± 2.83	18.5 ab ± 00	48.64 b ± 2.6	41.03 b ± 19.46	47.0 b ± 10.5
SLch6	22 ab ± 2.83	25.5 a ± 0.76	50.00 ab ± 1.57	42.15 ab ± 8.19	63.1 b ± 5.0
SLch14	15.4 b ± 5.44	18.8 ab ± 4.42	55.15 ab ± 14.34	41.18 b ± 10.72	60.0 a ± 5.0

Different letters (a, b, and c) indicate significantly different groups (p < 0.05) according to Tukey’s test.

). Statistical analysis using one-way ANOVA followed by Tukey’s post hoc test (p < 0.05) revealed significant differences among some strains. For chloroform, strains SCC1-13 (30.4% ± 7.58^{^a^}), SCC1-15 (28.6% ± 5.05^{^a^}), and SLch6 (25.5% ± 0.76^{^a^}) formed a group with the highest adhesion values, significantly differing from SCC1-2 (12.0% ± 5.66^{^b^}), which showed the lowest adhesion. Other strains displayed intermediate adhesion levels not significantly different from either group.

Regarding adhesion to ethyl acetate, the highest adhesion was observed in SCC1-8 (34.5% ± 4.88^{^a^}), which was significantly higher than SCC1-13 (0.0% ± 0.0^{^b^}), indicating distinct surface properties. Most strains clustered into groups reflecting low to moderate adhesion values, consistent with predominantly acidic and electron-acceptor surface characteristics.

Notably, SCC1-13 exhibited a significantly greater affinity for chloroform compared to ethyl acetate (30.4% vs. 0%, p < 0.05), suggesting a basic or electron-donor surface nature, as previously reported [37].

Our results are in agreement with previous studies by Kos et al. [24], who reported similar trends for Lactobacillus acidophilus M92 and E. faecium L3, with high adhesion to chloroform (36.06% and 61.21%, respectively) and no adhesion to ethyl acetate. Similarly, Xu et al. [38] observed high adhesion to chloroform in L. brevis (52.9%), Leuconostoc mesenteroides (51.2%), and L. rhamnosus GG (47.7%), while strains such as L. acidophilus ADH (22.6%) and Pediococcus acidilactici (25.8%) showed lower affinities. In contrast to chloroform, bacterial adhesion to ethyl acetate was generally lower, ranging between 5.1% and 16.9%.

Comparable findings were reported by Singh et al. [39] for various L. reuteri strains, with adhesion to chloroform ranging from 32.86% to 80.45%, and relatively lower values for ethyl acetate, aligning with our results. Other authors have also described variable adhesion capacities, typically between 5% and 47% [40].

3.4.2. Adhesion to Gastric Mucin and STC-1 Cells

The adhesion and colonization of probiotic bacteria within the host gastrointestinal tract are considered essential for exerting their beneficial effects. This includes preventing washout by intestinal peristalsis [41], promoting colonization [42], stimulating the host immune system [43], and providing antagonistic activity against enteropathogens [44].

The selected Enterococcus faecium strains were evaluated for their adhesion to gastric mucin at different growth phases (Figure 1). All the strains exhibited strong adhesion to mucin, with rates exceeding 60% throughout all the stages of growth. However, a decline in adhesion was observed during the stationary phase (gray), likely due to nutrient depletion and the accumulation of metabolic byproducts.

The adhesion capacity of the ten E. faecium strains to STC-1 epithelial cells was assessed in vitro. Overall, the strains demonstrated relatively low adhesion levels. Only two strains—SCC1-33 and SLch6—exhibited a more pronounced ability to adhere to epithelial cells, with adhesion values of 7.8 × 10³ and 4.2 × 10³ CFU/mL, respectively.

These results are in agreement with previous reports that describe moderate adhesion abilities in probiotic Enterococcus strains [45,46]. These results are consistent with previous studies on Enterococcus species: [47,48] described moderate adhesion levels in E. faecium and E. faecalis isolates, typically ranging from around 10³ to 10⁵ CFU/mL on Caco-2 cells.

While the adhesion levels observed here are lower than those typically reported for certain other genera, such as Bacillus or Bifidobacterium [49,50], this comparison is intended only to contextualize the diversity in adhesion behaviors among probiotics and not as a direct equivalence due to genus-specific differences in surface structures and adhesion mechanisms.

3.5. Auto-Aggregation and Co-Aggregation

Auto-aggregation and co-aggregation are important functional properties of probiotic bacteria, as they contribute to colonization and persistence within the gastrointestinal tract. These characteristics can enhance the ability of probiotics to form biofilms, adhere to epithelial surfaces, and compete with pathogenic microorganisms, thereby reducing their likelihood of being eliminated through intestinal transit [51]. The auto-aggregation and co-aggregation abilities of the selected probiotic strains are presented in Table 2. Among the ten Enterococcus faecium strains tested, six exhibited a high auto-aggregation rate, with values exceeding 60% after 24 h. According to Collado et al. [23], auto-aggregation values above 50% are generally considered indicative of strong self-aggregation ability.

All the strains also demonstrated strong co-aggregation with Saccharomyces cerevisiae, with the highest rates observed at 20 °C. These results suggest that temperature can influence co-aggregation behavior, potentially affecting adhesion dynamics. In comparison, Ayyash et al. [34] reported lower co-aggregation levels for the Enterococcus and Streptococcus strains isolated from camel milk when co-incubated with pathogenic bacteria at both 20 °C and 37 °C. According to Jensen et al. [52], bacterial adhesion is a multifactorial process, not solely attributed to a single component. It involves electrostatic interactions, hydrophobic interactions, and specific bacterial surface structures, all contributing to the overall adhesion and aggregation process.

3.6. Survival in Simulated In Vitro Digestion

The ability of probiotic strains to withstand the harsh conditions of the gastrointestinal tract (GIT) is essential for their therapeutic efficacy, as their health benefits rely on surviving and reaching the intestine in adequate numbers. In this study, selected Enterococcus faecium strains were exposed to simulated gastric and intestinal digestion to assess their viability under these stress conditions. As illustrated in Figure 2, strains SCC1-2 and SCC1-7 demonstrated the capacity to multiply in simulated intestinal fluid, whereas SCC1-15 and SLch6 showed survival rates exceeding 50%. Conversely, three strains exhibited low survival rates ranging from 11% to 37%, and two strains displayed poor survival, with less than 5% viability. These results are consistent with previous findings by Nascimento et al. [53], who reported limited survival of E. faecium strains SJRP20 and SJRP65 under similar conditions. Although these strains provide valuable comparative references, survival rates of probiotic E. faecium vary considerably depending on strain-specific characteristics and experimental setups, as highlighted by other studies.

3.7. Antagonistic Effect

The antagonistic activity of probiotic strains, especially their ability to inhibit pathogens in the GIT, is a key trait for their potential as probiotics. The antagonistic activity of the ten Enterococcus faecium strains was tested against various pathogens (Figure 3). All the strains demonstrated significant antagonistic effects against the tested pathogens, with varying intensities between strains. However, the SCC1-13 strain did not inhibit the growth of Micrococcus luteus. Several studies have reported the antibacterial activity of Enterococcus species. For instance, Enterococcus strains have an inhibitory spectrum that includes not only Gram-positive pathogens such as Listeria monocytogenes, Staphylococcus spp., and Clostridium spp. [54], but also Gram-negative bacteria, fungi, and yeasts [55]. E. faecium LCW 44, isolated from camel milk, exhibited a broad antibacterial spectrum, showing inhibitory activity against several Gram-positive strains from the genera Clostridium, Listeria, Staphylococcus, and Lactobacillus [56].

3.8. Safety Evaluation of E. faecium Strains

Antibiotic resistance in Enterococcus species is a major concern in the medical field, as resistance genes are frequently associated with plasmids or transposons, increasing the risk of horizontal gene transfer within microbial communities, especially in the gut [57]. In this study, the antibiotic susceptibility of the selected Enterococcus faecium strains was evaluated using the disk diffusion method (

Table 3. Antibiotic resistance and susceptibility of the studied strains of E. faecium.

Strains	T30	R30	V30	E15	A10	K1000
SCC1-2	S	R	S	R	S	S
SCC 1-6	S	R	S	S	S	S
SCC 1-7	S	R	S	S	S	S
SCC 1-8	S	R	S	S	S	S
SCC 1-13	S	R	S	S	R	S
SCC 1-15	S	R	S	S	S	S
SCC 1-24	S	R	R	S	S	S
SCC 1-33	S	R	S	S	S	S
SLC ch6	S	R	S	S	S	S
SLC ch14	S	R	S	S	S	S

T30: tetracycline; R30: rifampicin; V30: vancomycin; E15: erythromycin; A10: ampicillin; K1000: kanamycin. (R): resistant, (S): susceptible

). Inhibition zone diameters were interpreted in accordance with CLSI standards (M100, [58]). All the strains showed susceptibility to tetracycline, vancomycin, erythromycin, ampicillin, and kanamycin, while resistance to rifampicin was observed. It is important to note that phenotypic susceptibility does not always correspond to the absence of resistance genes. Therefore, these results should be interpreted cautiously. Future work will include molecular screening (e.g., PCR) for known antibiotic resistance genes to better assess the safety of the strains with respect to their probiotic use.

Comparatively, De Souza et al. [59] reported resistance to vancomycin in Lactobacillus casei and Lactobacillus fermentum strains, while Le Blanc [60] described E. faecium isolates resistant to glycopeptide antibiotics such as vancomycin and teicoplanin. The antibiotic susceptibility profile of strain SLCh6 is consistent with E. faecium strains isolated from food sources that comply with international safety guidelines [61].

In addition, the cytotoxicity test on STC-1 cells revealed no significant adverse effects for any of the strains tested, supporting their safety for potential probiotic application.

4. Conclusions

The present study demonstrates that camel milk can serve as a valuable source of potential probiotic candidates. Among the isolates, Enterococcus faecium SLCch6 exhibited promising characteristics for future applications in both the food industry and therapeutic contexts. This strain showed notable resilience to simulated gastrointestinal conditions, high auto-aggregation and co-aggregation abilities, strong adhesion to gastric mucin, and acceptable adhesion to epithelial cells—traits indicative of a robust capacity for survival and colonization within the gastrointestinal tract.

In addition, E. faecium SLCch6 exhibited significant antagonistic activity against several pathogenic microorganisms. Its susceptibility to commonly used antibiotics suggests a favorable preliminary safety profile. However, as phenotypic traits do not necessarily reflect genotypic characteristics, further genomic analysis is required to confirm the absence of transferable antibiotic resistance genes. Moreover, in accordance with the internationally accepted definition of probiotics—“live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”—it is important to emphasize that E. faecium SLCch6 should be regarded as a potential probiotic. Comprehensive in vivo studies and detailed safety evaluations are essential to confirm its health benefits and establish its suitability for clinical or commercial use.