The search for natural alternatives to chemical preservatives and antibiotics has gained significant traction in the food industry, particularly within the dairy sector. Lactic acid bacteria (LAB) are central to this movement due to their GRAS (Generally Recognized As Safe) status and their ability to produce a diverse array of antimicrobial compounds effective against pathogens (Papadimitriou et al., 2016; Doukaki et al., 2024). Species of the genus Lactobacillus are especially noted for synthesizing bioactive metabolites, such as organic acids, antimicrobial peptides and bacteriocins, which inhibit spoilage organisms and enhance the microbiological stability of fermented dairy products (Zhang et al., 2023). Furthermore, there is growing interest in postbiotics, specifically cell-free supernatants (CFS), due to their inherent stability and their utility in applications where introducing viable microbial cells is not desired (Zółkiewicz et al., 2020).
         
Honey is a unique natural matrix combining intrinsic antimicrobial properties with specialized microbial biodiversity. Beyond its well-documented bioactive compounds, studies show that honey is a natural reservoir of lactic acid bacteria (LAB) adapted to extreme ecological conditions, which produce microbiologically active metabolites (Adriani et al., 2024; Olofsson and Vásquez, 2008; Meradji et al., 2023; Saini et al., 2020).
       
In Algeria, research indicates that Lactobacillus strains isolated from local honeys possess significant antibacterial activity. Homrani et al., (2019) reported that isolates from raw Algerian honey notably inhibited Gram-negative bacteria. Similarly, Radja et al., (2022) confirmed the antimicrobial effectiveness of Lactobacillus strains from the Mostaganem region against various foodborne pathogens. Despite these advances, little is known regarding the antibacterial activity of cell-free supernatants (CFS) produced by Lactobacillus strains from the Ouarsenis region, a zone characterized by rich floral diversity and unique environmental conditions. Therefore, this study aims to isolate and characterize Lactobacillus strains from Ouarsenis honey (Algeria) and evaluate the antibacterial potential of their cultures and CFS against selected pathogens. Additionally, this work seeks to explore the application of these microbial metabolites as natural biopreservatives in food matrices.

Collection of honey samples
 
 A total of nine raw honey samples (n = 9), collected during the apicultural production period, from June to August 2022, in the Ouarsenis region (northwestern Algeria), were obtained directly from beekeepers under controlled aseptic conditions. Samples were, stored in sterile containers and kept at -20°C until analysis. Information on floral origin was provided by the beekeepers based on the location of the apiaries (Table 1).


       
All experimental analyses were carried out at the Laboratory of Sciences and Techniques of Animal Production, Department of Agronomy, Faculty of Natural and Life Sciences, Abdelhamid Ibn Badis University of Mostaganem, 27000, Algeria.
 
Isolation and biochemical screening of Lactobacilli
 
Isolation of Lactobacilli was carried out according to Homrani et al., (2019) and Radja et al., (2022). Briefly, ten grams (10 g) of each raw honey sample were aseptically weighed and homogenized with 90 mL of sterile peptone saline solution (NaCl 0.9%, peptone 0.1%), containing 0.1% Tween 80.  One milliliter of the homogenate was inoculated into 9 mL of de Man, Rogosa and Sharpe (MRS) broth and incubated at 30°C for 24 h. Decimal dilutions (10^-1 to 10^-5) were prepared in sterile peptone water and 100 µL of each dilution were spread onto selective agar media (MRS, MRS supplemented with 0.8% CaCO₃ and MRS supplemented with 0.1% L-cysteine and Rogosa agar). Plates were incubated at 37°C under anaerobic conditions for 72 h.
       
Only Catalase-negative (not releasing gas bubbles in the presence of H2O), Gram-positive, rod-shaped colonies were selected, purified on MRS agar and stored at -20°C in MRS broth with 20-30/ % glycerol for further analyses.
       
The selected isolates were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) using the Bruker MALDI Biotyper system, following the manufacturer’s recommended protocol. Briefly, fresh part of the cultivated colony on MRS agar was smeared on and immediately covered with 1 μL of matrix solution. The plate was then analyzed with the MALDI-TOF apparatus and mass spectra were compared with reference spectra in the manufacturer’s database. Identification confidence levels were interpreted as follows: high-confidence (+++), low-confidence (+) and no reliable identification (-).
 
Indicator bacterial strains
 
For the assessment of antimicrobial activity, three pathogenic strains: Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae were used as indicator strains. These strains were obtained from the private laboratory and were originally isolated from patients with urinary tract infections. Identification of the bacteria was carried out through biochemical testing using the API 20 system.
 
Antibacterial activity of Lactobacillus isolates by agar spot assay
 
The antibacterial activity of Lactobacillus isolates was evaluated using the agar spot assay, according to the method described by Fleming et al., (1975). Overnight (18-24 h) cultures of Lactobacillus grown in MRS broth were spotted onto MRS agar plates and incubated at 37°C for 18 h under anaerobic conditions. Overnight (18-24 h) cultures of the indicator strains, grown in nutrient broth, were then incorporated into soft nutrient agar and poured over the plates containing the Lactobacillus colonies. After aerobic incubation at 37°C for 24 h, antibacterial activity was assessed by measuring the diameters of inhibition zones around the spots, expressed in millimeters.
 
Evaluation of antibacterial properties of cell-free supernatants from selected lactobacilli
 
The antibacterial activity of CFS from Lactobacillus isolates was assessed using the agar well diffusion method, following Barefoot and Kaenhammer (1983). The isolates were grown in MRS broth at 30°C for 18-24 h, after which CFS were obtained by centrifugation (6,000 rpm, 20 min, 4°C) and sterile filtration. Wells of 5 mm diameter were filled with 100 µL of CFS on nutrient agar plates previously inoculated with the indicator strains and incubated at 37°C for 18 h. Antibacterial activity was determined by measuring the inhibition zones around the wells.
         
The CFS exhibited significant inhibitory effects against pathogens such as Escherichia coli, Staphylococcus aureus and Salmonella spp., indicating the presence of antimicrobial compounds such as organic acids and bacteriocins (Hacıoğlu and Türkyılmaz, 2024; Hussein et al., 2025).
 
Data analysis
 
The diameters of the inhibition zones were measured in millimeters and expressed as mean±standard deviation (SD) from two independent replicates. Mean and standard deviation calculations were performed using Microsoft Excel (Microsoft Corporation, USA). No inferential statistical analysis was applied.
       
The antibacterial activity of the strains was classified based on the inhibition zone diameters, according to interpretative criteria commonly used in recent studies on lactic acid bacteria isolated from honey: weak activity for diameters <10-12 mm, moderate activity for diameters between 10-20 mm and strong activity for diameters ≥20 mm (Meradji et al., 2023; Roy and Mandal, 2024; Zamri et al., 2023).

Isolation and screening of lactobacilli from ouarsenis raw honey
 
Following pre-enrichment in MRS broth and plating on four selective media, over 150 colonies were screened for Gram reaction, morphology and catalase activity. Fifty isolates (n=50) were confirmed as presumptive Lactobacillus genus based on their catalase negative Gram positive and bacilli form (Table 2).


       
The preliminary isolation and characterization of LAB from raw honey sample collected in the Ouarsenis region revealed that the isolates were successfully cultivated on various selective media (MRS, MRS-CaCO₃, MRS-cysteine and Rogosa medium), which promoted the growth of LAB while limiting unwanted microorganisms.
       
All analyzed isolates were Gram-positive, catalase-negative and exhibited a rod-shaped morphology, typical characteristics of the genus Lactobacillus. These findings are consistent with previous reports of lactobacilli isolated from raw honey and beekeeping environments, confirming the relevance of these criteria for preliminary phenotypic identification (Meradji et al., 2023).
 
Identification of Lactobacillus isolates by MALDI-TOF MS
 
MALDI-TOF MS analysis (Table 3) provided a rapid, high-throughput identification of eleven LAB isolates from Ouarsenis honey, successfully discriminating four distinct species (Limosilactobacillus fermentum, Lactobacillus plantarum, Apilactobacillus kunkeei and Fructobacillus fructosus) with high confidence scores (≥2.0). These findings highlight the diversity of the honey microbiota, featuring both typical honeybee-associated, fructophilic bacteria and generalist LAB. 


       
However, the analysis encountered limitations in distinguishing closely related taxa. For example, isolate Lb29 displayed ambiguous, close-match profiles between L. plantarum and L. pentosus, reflecting known limitations of MALDI-TOF in resolving species within the Lactobacillus plantarum group, which often requires more than just mass spectral protein profiles for precise identification. Similarly, the low-confidence score for Lb34 (L. fermentum), despite a high-confidence secondary match, underscores potential database gaps or high strain-level variability in ribosomal proteins. The inability to identify isolates Lb18 and Lb5 (scores < 1.7) further indicates that these may represent rare or novel species not well-represented in current taxonomic libraries. 
       
To reconcile these ambiguous results and ensure species-level precision, complementary molecular approaches are necessary. 16S rRNA gene sequencing or, ideally, Multi-Locus Sequence Typing (MLST) should be employed to confirm identification for low-score isolates, allowing for a deeper understanding of the ecological roles of these specific honey-derived strains.
       
The Lactobacillus profiles identified in this study align with the microbial signatures frequently documented in honey and related apicultural matrices. Notably, Apilactobacillus kunkeei emerged as a hallmark species; it is widely regarded as a quintessential fructophilic lactic acid bacterium (FLAB) within apicultural niches. This dominance is likely driven by its specialized metabolic pathways and its high prevalence within the honeybee gut microbiota (Takatani and Endo, 2021). Furthermore, the detection of Lactobacillus plantarum and Limosilactobacillus  fermentum corroborates previous findings where these species demonstrated robust adaptation to the high osmotic pressure characteristic of honey (Iorizzo et al., 2020; Meradji et al., 2023).
         
In a comparative context, Abadi et al., (2023) observed a similar predominance of L. plantarum in Iranian honey, though they also reported a minor presence (7.69%) of L. rhamnosus and L. acidophilus. Interestingly, our findings suggest a more complex microbial diversity than previously reported for Algerian honeys. For instance, earlier investigations by Homrani et al., (2019) in the regions of Mostaganem, Medea and Souk Ahras primarily isolated L. plantarum. Similarly, Radja et al., (2022) identified a recurring dominance of L. plantarum and L. pentosus in samples from Mostaganem.
       
However, our results echo the broader taxonomic spectrum observed by Meradji et al., (2023) in northeastern Algeria. Their study of honey and bee stomach samples identified more specialized species, including  Fructobacillus fructosus, Apilactobacillus kunkeei and members of the L. kullabergensis and L. kimbladii lineages. This increased diversity in our study may reflect localized variations in floral sources, environmental conditions, or enhanced detection methodologies compared to earlier regional surveys.
       
LAB in honey originate from several synergistic pathways. A significant fraction is derived from the honeybee’s gut microbiota; dominant Lactobacillaceae phylotypes are shed onto nectar during foraging and integrated into the honey during its maturation (Engel et al., 2016 ; Bonilla-Rosso and Engel, 2018). Furthermore, pollen serves as a vital microbial vector, introducing LAB into the hive during nectar processing and subsequent storage (Parichehreh et al., 2025). While the hive environment may offer additional microbial inputs, the harsh physicochemical profile of honey acts as a selective filter. Consequently, only the most resilient strains persist, often exhibiting a progressive decline in viability over time (Luca et al., 2024).
 
Antibacterial activity of lactobacillus isolates against selected pathogens
 
Table 4 details the antagonistic potential of various Lactobacillus cultures and their CFS against a panel of pathogenic indicators: Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. 


       
The whole-cell bacterial cultures displayed robust antibacterial profiles, with inhibition zones spanning from
10±0 mm to a substantial 39.5±0.71 mm (mean: 26.2± 0.9 mm). In the challenge against E. coli, Limosilactobacillus fermentum (Lb3) and Apilactobacillus kunkeei (Lb15) emerged as the most potent antagonists, yielding clear zones of 39.5±0.71 mm and 38.5±0.71 mm, respectively. These were followed by notable activity from Fructobacillus fructosus (Lb10, 35.5±0.71 mm) and Lactobacillus plantarum (Lb19, 29.5±0.71 mm). Conversely, more modest inhibitory effects were recorded for isolates Lb34, Lb29 and Lb23.
       
Regarding K. pneumoniae, the hierarchy of efficacy shifted slightly; F. fructosus (Lb10) proved most effective (38.5±0.71 mm), followed by L. plantarum (Lb19, 31.5± 0.71 mm). While A. kunkeei (Lb15) and L. fermentum (Lb3) maintained strong inhibitory roles, isolates Lb29, Lb34 and Lb23 demonstrated significantly more moderate to marginal suppression of this pathogen.
       
The antagonistic potential of the tested LAB against S. aureus revealed a spectrum of efficacy, with Lb15 (A. kunkeei), Lb10 (F. fructosus) and Lb19 (L. plantarum) emerging as the most potent inhibitors, yielding inhibition zones of 27±1.41 mm, 25.5±0.71 mm and 22±0.0 mm, respectively. In contrast, Lb34 (L. fermentum)  demonstrated   moderate suppression, while Lb23 (F. fructosus) exhibited the most constrained activity. The pronounced susceptibility of S. aureus relative to Gram-negative counterparts likely stems from the structural simplicity and higher permeability of the Gram-positive cell wall. This lack of an outer membrane facilitates the deep penetration of  LAB-derived metabolites, including organic acids (lactic and acetic), hydrogen peroxide and antimicrobial peptides, thereby disrupting cellular homeostasis more effectively (Chen et al., 2025; Zhang et al., 2023; Hussein et al., 2025).
       
Regarding the CFS, inhibitory diameters fluctuated between 12±0 mm and 24±0 mm, underscoring a strain-specific and pathogen-dependent antimicrobial profile. While Lb15 and Lb19 were particularly adept at curbing E. coli, Lb46 and Lb19 proved superior against K. pneumoniae. Notably, the most robust CFS action was recorded against S. aureus by Lb46 (24±0 mm) and Lb19 (20±0 mm). These findings reinforce the premise that the biocontrol efficiency of CFS is a multifaceted phenomenon governed by the unique metabolic repertoire of the LAB isolate and the specific defensive architecture of the target pathogen (Hernández-Figueroa et al., 2024 ; Shaaban et al., 2025 ; Aliouche et al., 2024). This variability suggests that specific LAB strains may be strategically selected for targeted application against either Gram-positive or Gram-negative clinical isolates.
       
This strain-dependent variability aligns with contemporary research on LAB sourced from honey and fermented matrices, which demonstrates that CFS harbor complex bioactive metabolites capable of potent pathogenic inhibition (Sisay et al., 2025; Roy and Mandal, 2024; Hussein et al., 2025; Boussif et al., 2026; Li et al., 2023; Shaaban et al., 2025; Aliouche et al., 2024; Chen et al., 2025; Hernández-Figueroa et al., 2024). Such diversity underscores that antimicrobial efficacy is not a generic trait of the genus but a specialized characteristic of individual strains. Consequently, the meticulous selection of high-performing LAB isolates is paramount for optimizing biotechnological and food-grade applications. These results further validate the promise of honey-derived LAB as robust candidates for postbiotic development or as functional adjunct cultures designed to fortify the microbiological integrity and shelf-life of food systems, particularly within the realm of fermented dairy products (Boussif et al., 2026).

Beyond its reputation as a regional delicacy, raw honey from the Ouarsenis mountains functions as a sophisticated ecological reservoir for bioactive Lactobacillus strains possessing transformative therapeutic potential. This study reveals that these autochthonous isolates exert potent antibacterial pressure against formidable clinical pathogens, specifically E. coli, K. pneumoniae and S. aureus, positioning them as formidable, nature-derived alternatives to traditional antibiotics. By characterizing this unique microbial profile, our findings elevate Ouarsenis honey from a mere functional food to a primary source of high-value probiotics with versatile applications in pharmaceutical formulation and food preservation. Ultimately, the biotechnological promise of these strains offers a strategic pathway to address the escalating global crisis of antimicrobial resistance, establishing the Ouarsenis region as a vital frontier for next-generation bioscientific innovation.

The authors sincerely thank the beekeepers of the Ouarsenis massif for their cooperation and for providing valuable information during the sample collection. They also express their gratitude to the University of Mostaganem, Department of Agronomy, as well as to the agricultural services of Tissemsilt province for their technical and logistical support.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare no conflict of interest.