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Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Genotypic Mapping and Functional Diversity of Lactic Acid Bacteria in Traditional Cheeses of the Central Algerian Steppe

I
I. Benzekri1,*
https://orcid.org/0009-0007-5697-0304
A
A. Cheriguene1
https://orcid.org/0000-0002-2618-8457
F
F. Chougrani2
https://orcid.org/0009-0005-3754-2940
A
A.A. Dahou2
https://orcid.org/0000-0001-6640-3169
A
A. Bekihal2
https://orcid.org/0000-0001-8372-6525
1Laboratory of Bioeconomy, Food Safety and Health, Faculty of Natural Science and Life, Abdelhamid Ibn Badis University, 27000 Mostaganem, Algeria.
2Laboratory of Sciences and Techniques of Animal Production, Faculty of Natural Science and Life, Abdelhamid Ibn Badis University, 27000 Mostaganem, Algeria.

ABSTRACT

Background: In Algerian steppe cheeses such as Jben and Klila, indigenous lactic acid bacteria (LAB) drive spontaneous fermentation, leading to rapid acidification, protein degradation and the production of flavor-enhancing compounds, including exopolysaccharides (EPS), as well as antimicrobial metabolites.

Methods: Ten LAB strains isolated from artisanal Jben and Klila cheeses of the Algerian steppes were identified using 16S rRNA sequencing. Their key functional traits, such as acidification capacity, proteolytic activity and exopolysaccharide-mediated texturization, were evaluated to select promising starter cultures for the preservation of traditional dairy products.

Result: Indigenous LAB dominate Jben and Klila cheeses, with species such as L. plantarum and E. faecium in Jben and L. pentosus and L. brevis in Klila, reflecting distinct metabolic roles. These strains combine rapid acidification, variable proteolytic activity and, in some cases, exopolysaccharide production, enhancing texture. Their genetic and functional diversity makes them strong candidates for developing multi-strain starter cultures that can standardize fermentation while preserving traditional characteristics.

INTRODUCTION

Traditional Algerian dairy products, including Jben, Klila and Bouhezza, constitute valuable components of the country’s cultural heritage and local economy. These cheeses are the result of long-standing artisanal practices developed in rural and steppe areas to ensure the preservation and valorization of milk under traditional conditions (Benamara et al., 2022; Leksir et al., 2019). 
       
In the southern and High Plateau regions of Algeria, Jben and Klila are highly appreciated for their nutritional value and their importance in traditional food systems. Jben is a fresh cheese obtained through acid coagulation, while Klila is a dried, shelf-stable product well suited to arid environments due to its reduced moisture content. Both products are produced through natural fermentation processes involving indigenous lactic acid bacteria (LAB), which play a key role in developing their characteristic sensory properties and ensuring product safety (Boumediene et al., 2024; Benamara et al., 2022; Leksir et al., 2019). 
       
LAB are essential in milk fermentation processes, where they drive acid production, protein breakdown and the synthesis of flavor compounds and exopolysaccharides, while simultaneously limiting the growth of undesirable microorganisms. The microbial composition of these traditional cheeses has been partly characterized: Jben is commonly associated with species such as Lactococcus lactis, Lactiplantibacillus plantarum and Leuconostoc spp., whereas Klila is typically dominated by more acid-resistant bacteria, including Lactiplantibacillus plantarum and Lactobacillus acidophilus. Some of these microorganisms are known to improve product quality and may also contribute to enhanced microbiological safety (Azzouz et al., 2025a; Bendimerad et al., 2024; Benamara et al., 2022; Bouchibane et al., 2022; Ketrouci et al., 2021). 
       
Although advances have been made in the identification of microbial communities, the functional profiling of indigenous lactic acid bacteria (LAB) from traditional Algerian cheeses remains insufficient, particularly regarding their technological performance and suitability as starter cultures. In particular, comprehensive studies evaluating their acidifying, proteolytic and texturizing capacities in combination are still scarce.
       
In this context, the present study focused on the isolation and identification of LAB from traditional Jben and Klila cheeses using 16S rRNA gene sequencing. In addition, their technological traits, including acidification ability, proteolytic activity and exopolysaccharide (EPS) production, were investigated. The objective was to select promising strains with potential application as starter cultures, thereby contributing to the preservation and valorization of the unique properties and cultural heritage associated with these traditional dairy products.

MATERIALS AND METHODS

Sampling and isolation
 
This work was conducted at the Laboratory of Sciences and Techniques of Animal Production (LSTPA), in collaboration with the Laboratory of Bioeconomy, Food Safety and Health (LBSAS), Faculty of Natural Sciences and Life, Abdelhamid Ibn Badis University of Mostaganem (Algeria), over the period from January 2024 to December 2025. A total of five samples of traditional cheeses (Jben and Klila), produced from raw cow’s milk, were obtained from artisanal producers in the Djelfa region. LAB isolation was performed by homogenizing the samples in a tryptone-salt solution, followed by cultivation on MRS and M17 agar media at 37°C and 45°C. Colonies exhibiting typical LAB characteristics (Gram-positive and catalase-negative) were selected and subsequently maintained either by periodic subculturing at 4°C or by storage at -20°C in glycerol for long-term preservation (Bouchibane et al., 2023; Bounaama et al., 2022).
 
Genotypic characterization
 
Molecular identification of the purified LAB isolates was carried out at the Gene Life Sciences (GLS) Laboratory, University of Sidi Bel-Abbès. Genomic DNA was extracted using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies, Malaysia) and its quality and concentration were assessed using a NanoDropTM One spectro photometer (USA). Amplification of the 16S rRNA gene was performed using the universal primers 27F (5’-AGA GTT TGA TCC TGG CTC AG-32 ) and 1387R (3’-GGG CGG WGT GTA CAA GGC-32 ), following the method described by Lane (1991). PCR reactions were prepared in a final volume of 50 µL, containing 25-50 ng/µL of template DNA, 0.3 µM of each primer, 1.5 µM MgCl‚  and 1.25 U of Hot Start Taq polymerase (Solis Biodyne, Estonia). Amplification was conducted using a SimpliAmp thermocycler (Applied Biosystems, USA). The thermocycling program consisted of an initial denaturation step at 94°C for 12 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min 40 s, with a final extension step at 72°C for 7 min.
       
PCR amplification products were examined by electrophoresis on 1.5% agarose gels, followed by staining and visualization under ultraviolet illumination. The resulting amplicons were then purified and subjected to bidirectional sequencing using the BigDye Terminator v3.1 kit. After thermal cycling, purification was carried out by ethanol precipitation and sequence fragments were analyzed by capillary electrophoresis using a 3730 XL Genetic Analyzer. The obtained sequences were edited and assembled using CHROMAS PRO software and taxonomic identification was performed through comparison with reference sequences in the NCBI database using the BLAST algorithm. Phylogenetic relationships were subsequently inferred using MEGA12 software based on the Neighbor-Joining method (Edis et al., 2025). 
 
Technological characterization
 
pH Kinetics and acidifying activity
 
Acidifying activity was assessed by measuring pH and titratable acidity in 10% skimmed milk inoculated at 1% and incubated at 30-37°C. Titratable acidity was determined by NaOH titration and expressed in Dornic degrees (°D), calculated (Dahou et al., 2021).
 
(°D) = VNaOH × 10
 
Where,
1/°D corresponds to 0.1/ g of lactic acid per liter of milk.
 
Proteolytic activity
 
Proteolytic activity was assessed using PCA medium supplemented with 10% skimmed milk. Sterile Whatman discs inoculated with LAB cultures were placed on the medium and incubated at 30-37°C for 24-72 h. The appearance of a clear zone around the discs indicated proteolytic activity (Bekihal et al., 2025).
 
Assessment of texturizing ability
 
This assessment was carried out at the Food Microbiology Laboratory of Istanbul Technical University (Turkey). EPS production was screened on modified BHI agar following anaerobic incubation at 30°C, where viscous and glossy colonies were considered positive. EPS was subsequently extracted and quantified by growing the isolates in modified BHI broth, followed by ethanol precipitation, TCA deproteinization, dialysis (12-14 kDa) and lyophilization (Ispirli et al., 2025). 
 
Statistical analysis
 
All experiments were conducted in triplicate (n = 3) and results are presented as mean ± standard deviation. Reproducibility was confirmed by a coefficient of variation below 5%. Data were analyzed using one-way ANOVA with IBM SPSS Statistics 25, followed by Student’s t-test. Statistical significance was set at p<0.05.

RESULTS AND DISCUSSION

Sampling and isolation
 
In this study, ten LAB isolates from two traditional cheeses of the Djelfa province (Jben and Klila) were collected from the localities of Aïn Oussera, Djelfa and Birine. The purified isolates were initially screened for Gram-positive and catalase-negative traits to confirm their identity as LAB (Fig 1).

Fig 1: Sample of Jben (a), Colony morphology of LAB isolate in MRS (b) and M17 (c), agar medium, Microscopic of LAB isolate (d)


       
Fig 1 shows the various stages of LAB observation. Colonies grown on MRS and M17 media were circular, smooth and ranged from whitish to cream in color, typical of LAB, indicating their adaptation to mesophilic conditions. Microscopic examination revealed cocci- or rod-shaped cells arranged in clusters or chains, characteristic of LAB commonly present in fermented cheeses. These results confirm the dominance of LAB and demonstrate their morphological diversity, supporting their potential for use in developing starter cultures.
 
Genotypic characterization
 
The isolation and identification results presented in Table 1 show that traditional Jben and Klila cheeses from the Djelfa region contain a diverse indigenous LAB community, primarily composed of Enterococcus and lactobacilli from the plantarum/brevis group. Ten isolates were identified through 16S rRNA gene sequencing as members of Enterococcus, Lactiplantibacillus and Levilactobacillus. This combined phenotypic (Fig 1) and molecular approach (Table 1) is commonly employed to characterize indigenous LAB in traditional cheeses and the assignment of accession numbers (PX736099–PX736107, PX737322) ensures traceability and facilitates their future use, as reported by Azzouz et al., (2025b) and Bouchibane et al., (2023).

Table 1: Source and region of the LAB species identified, including their accession numbers.


       
The species distribution shows cheese-specific patterns, with E. faecium and L. plantarum dominating in Jben and L. pentosus and Lev. brevis primarily associated with Klila. This pattern aligns with previous studies on traditional Mediterranean and Algerian cheeses, as well as dairy products from the Black Sea region and Morocco. These results confirm that Jben and Klila host characteristic LAB species, underscoring their potential for the development of indigenous starter cultures with both technological and probiotic applications (Azzouz et al., 2025b; Bouchibane et al., 2023).
       
The phylogenetic tree (Fig 2) displays distinct clustering of isolates by species, showing high similarity to reference strains and confirming their identification via 16S rRNA gene analysis as members of the genera Enterococcus, Lactiplantibacillus and Levilactobacillus. Such concordance between 16S rRNA-based phylogenetic trees and reference taxonomy is widely used to validate bacterial identification, including LAB. The short branch lengths and tight clustering indicate low genetic divergence and strong phylogenetic relatedness, reflecting relatively homogeneous populations (Al-shammary et al., 2025).

Fig 2: Phylogenetic tree of 16S ribosomal RNA gene partial sequence of LAB species identified, including their accession numbers.


 
Technological characterization
 
pH kinetics and acidifying activity
 
All isolates showed a gradual decrease in pH accompanied by a corresponding increase in titratable acidity (°D) over 24 h, confirming their fermentative activity (Fig 3). Initial measurements were consistent (pH ≈6.70; acidity ≈19°D), reflecting standardized experimental conditions. By 6 h, a moderate acidification phase was observed, with pH values between approximately 5.75 and 6.15 and acidity rising to 24-36°D. After 24 h of incubation, pH values ranged from 4.02 to 5.80, while titratable acidity reached 33-83°D (Table 2).

Fig 3: Kinetics of pH and titratable acidity of LAB isolates.



Table 2: Technological activities of LAB Isolated from Jben and Klila.


       
Statistical analysis revealed highly significant differences among isolates (p<0.01), confirming variability in acidifying performance. Strains IB4 (JDj03) and IB2 (JDj02) showed the highest acidification capacity (pH ≈4.02-4.16; acidity ≈80-83°D), followed by IB7 (JDj04), IB10 (JDj12) and IB5 (LO23), which also exhibited strong acidifying activity (acidity ≥66°D). In contrast, IB3 (JO09) and IB1 (JO01) displayed significantly weaker acidification (p<0.05), with pH values above 5.2 and acidity below 40°D, while IB6 (JB01), IB8 (CDz09) and IB9 (LB10) showed intermediate profiles.
       
Based on these results, isolates can be classified into three functional groups: strongly acidifying strains (pH drop ≥ 2 units; acidity ≥66°D), moderately acidifying strains and weakly acidifying strains. A clear inverse relationship between pH and acidity was observed, reflecting lactose conversion into lactic acid.
       
The acidification kinetics observed followed typical lactic fermentation patterns, characterized by a decrease in pH and an increase in titratable acidity within 24 h. Isolates such as JDj03, JDj02 and LO23 exhibited strong acidifying capacity, confirming their suitability as starter cultures, as rapid acidification enhances milk coagulation and microbial safety. Moderately acidifying strains (CDz09, LB10, JB01) may serve as adjunct cultures, while weak acidifiers (JO01, JO09) were more likely involved in flavor development and maturation, as reported by Durango Zuleta et al. (2023) and Sesín et al. (2023).
       
These findings are supported by the significant pH reduction and acidity values exceeding 60°D observed in active LAB. Overall, the functional diversity among isolates supports the development of multi-strain starter cultures combining complementary properties to improve cheese quality and standardization while preserving traditional characteristics, in agreement with previous studies reported by Grujović et al. (2024), Sesín et al. (2023) and Coelho et al., (2022).
             
Proteolytic activity
 
Proteolytic activity, measured by halo diameter (mm), reflects protein hydrolysis and protease production (Table 2). Halo sizes ranged from 0 to 36 mm, indicating substantial variability among isolates. Statistical analysis showed highly significant differences (p<0.001), confirming pronounced functional heterogeneity. While the high coefficient of variation highlights inter-strain differences, low standard deviations indicate good reproducibility of the measurements. Based on halo diameters, isolates were categorized into three functional groups:
• Super-proteolytic (≥ 30 mm): JO01 (31 mm), JDj03 (36 mm) and LO23 (36 mm), corresponding to the highest statistical group a (p<0.05).
• Moderately proteolytic (10-29 mm): JDj02 (20 mm), JB01 (23 mm) and CDz09 (10 mm), falling into intermediate groups b and c.
• Weakly or non-proteolytic (< 10 mm or 0 mm): JO09, JDj04, LB10 and JDj12, forming a homogeneous group d.
       
The high activity observed in super-proteolytic strains reflects a strong capacity for casein degradation, promoting peptide formation and flavor development. These strains therefore represent excellent candidates for accelerating cheese ripening and enhancing their bioactive properties, particularly in fresh or ripened cheeses, as reported by Novak et al., (2021). Isolates with moderate activity play an essential intermediate role by ensuring controlled protein hydrolysis. They help limit bitterness and stabilize the rheological properties of the curd, acting as “metabolic bridges” within the microbial ecosystem. In contrast, weakly or non-proteolytic strains were mainly involved in rapid acidification of the medium, contributing to microbiological safety without directly participating in protein degradation, as described by Coelho et al., (2022).
       
This functional stratification, ranging from highly proteolytic to primarily acidifying strains, reflects a rational approach to starter culture selection. Combining these complementary profiles makes it possible to design optimized mixed cultures capable of synchronizing acidification, proteolysis and flavor development, thereby meeting the technological and sensory requirements of traditional cheeses, according to Coelho et al., (2022) and Novak et al., (2021).
 
Assessment of texturizing ability
 
The ability of the ten LAB isolates from Jben and Klila to produce exopolysaccharides was first screened qualitatively on sucrose-enriched modified BHI agar. Only three isolates, JO01 (IB1), LB10 (IB9) and JDj12 (IB10), displayed a clear viscous (“slimy”) phenotype, indicating active EPS synthesis. This observation is consistent with the common use of colony viscosity as a rapid indicator of EPS-producing strains.
       
Quantitative analysis performed under controlled conditions (30°C, 48 h, anaerobiosis) confirmed these results. After extraction, purification and lyophilization (Fig 4), EPS yields reached 0.69±0.25 g/L for IB1, 0.86±0.30 g/L for IB9 and 0.56±0.20 g/L for IB10 (Table 2), whereas no detectable production was observed for the remaining isolates. The overall production range (0-0.86 g/L) and the relatively high standard deviations highlight pronounced inter-strain variability, which was statistically significant (p<0.05). This heterogeneity reflects the presence of a limited number of efficient producers within a predominantly non-producing population.

Fig 4: From left to right: bacterial fermentation broth, centrifuged cell pellets, purified EPS after dialysis and finally, lyophilized EPS powder.


       
The calculated sugar-to-EPS conversion efficiency (based on 7.5 g sucrose/250 mL) ranged from 1.85% to 2.86%, indicating an active but non-optimized metabolic capacity for polysaccharide synthesis. Despite the absence of process optimization, these values suggest that the selected strains possess a favorable baseline for EPS production.
       
From a technological perspective, EPS-producing isolates are of particular interest due to their ability to improve the rheological properties of fermented dairy products. These polymers contribute to viscosity enhancement, water retention and structural stabilization of the matrix. In contrast, non-producing strains may still play complementary roles, such as acidification and microbial balance, supporting the concept of mixed starter cultures combining EPS-positive and EPS-negative strains to achieve both textural and microbiological benefits.
       
When compared with literature data, the yields obtained in this study can be considered relatively high for non-optimized systems. For example, the marine isolate MSD8 produced only 0.20 g/L under similar conditions (Abdel-monem et al., 2024), which is markedly lower than the values reported here. This difference suggests that isolates from traditional dairy environments may be naturally better adapted for EPS biosynthesis.
       
Nevertheless, higher production levels have been described for certain Enterococcus faecium strains. Under rich but non-optimized conditions, strains such as R114 and T52 (isolated from Kishk) reached 2.68 and 2.39 g/L, respectively (Rahnama Vosough et al., 2022), illustrating the strong influence of strain-specific metabolic potential. Furthermore, optimization strategies based on statistical designs (e.g., RSM or CCD) have enabled yields of approximately 2.5-3.2 g/L for strains such as R114, F58 and KT990028 (De Brito et al., 2024; Zanzan et al., 2023; Rahnama Vosough et al., 2021). In some cases, extreme production levels have been achieved, particularly for fish-derived strains such as MC13 and MC-5, reaching up to 11.6 and 16.5 g/L after optimization (Tilwani et al., 2021; Kanmani et al., 2013).
       
Overall, the EPS yields obtained for IB1, IB9 and IB10 fall within the upper range typically reported for LAB cultivated under non-optimized conditions. Although lower than those achieved after process optimization, these results demonstrate a strong intrinsic production capacity. This baseline performance provides a solid foundation for future improvement through bioprocess optimization and supports the potential application of these strains in food and biotechnological fields.

CONCLUSION

The microbial landscape of traditional Djelfa cheeses, Jben and Klila, represents a sophisticated ecological blueprint shaped by centuries of artisanal heritage. Our findings reveal a niche-specific distribution, where Lactiplantibacillus plantarum and Enterococcus faecium drive the fresh profile of Jben, while L. pentosus and Levilactobacillus brevis define the robust character of Klila. This genetic stratification is mirrored by a “technological division of labor”: rapid acidifiers provide microbial safety, while moderate acidifiers, proteolytic and EPS-producing strains orchestrate sensory complexity and the release of health-promoting bioactive peptides. These indigenous consortia do not merely represent local biodiversity; they provide a high-performance toolkit for the design of “terroir-driven” starter cultures. Utilizing these native strains offers a dual advantage: ensuring the industrial reproducibility of traditional textures and flavors while preserving the biological signature of Algerian dairy heritage.

ACKNOWLEDGEMENT

The present study was supported by the Faculty of Natural and Life Sciences, Abdelhamid Ibn Badis University. I would like to express my sincere gratitude to the staff of both research laboratories and to the DGRSDT for their valuable support in advancing scientific research in Algeria.
 
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.
 
Informed consent
 
This study does not involve any animal experimentation and no procedures related to the use of animals were conducted.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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    Genotypic Mapping and Functional Diversity of Lactic Acid Bacteria in Traditional Cheeses of the Central Algerian Steppe

    I
    I. Benzekri1,*
    https://orcid.org/0009-0007-5697-0304
    A
    A. Cheriguene1
    https://orcid.org/0000-0002-2618-8457
    F
    F. Chougrani2
    https://orcid.org/0009-0005-3754-2940
    A
    A.A. Dahou2
    https://orcid.org/0000-0001-6640-3169
    A
    A. Bekihal2
    https://orcid.org/0000-0001-8372-6525
    1Laboratory of Bioeconomy, Food Safety and Health, Faculty of Natural Science and Life, Abdelhamid Ibn Badis University, 27000 Mostaganem, Algeria.
    2Laboratory of Sciences and Techniques of Animal Production, Faculty of Natural Science and Life, Abdelhamid Ibn Badis University, 27000 Mostaganem, Algeria.

    ABSTRACT

    Background: In Algerian steppe cheeses such as Jben and Klila, indigenous lactic acid bacteria (LAB) drive spontaneous fermentation, leading to rapid acidification, protein degradation and the production of flavor-enhancing compounds, including exopolysaccharides (EPS), as well as antimicrobial metabolites.

    Methods: Ten LAB strains isolated from artisanal Jben and Klila cheeses of the Algerian steppes were identified using 16S rRNA sequencing. Their key functional traits, such as acidification capacity, proteolytic activity and exopolysaccharide-mediated texturization, were evaluated to select promising starter cultures for the preservation of traditional dairy products.

    Result: Indigenous LAB dominate Jben and Klila cheeses, with species such as L. plantarum and E. faecium in Jben and L. pentosus and L. brevis in Klila, reflecting distinct metabolic roles. These strains combine rapid acidification, variable proteolytic activity and, in some cases, exopolysaccharide production, enhancing texture. Their genetic and functional diversity makes them strong candidates for developing multi-strain starter cultures that can standardize fermentation while preserving traditional characteristics.

    INTRODUCTION

    Traditional Algerian dairy products, including Jben, Klila and Bouhezza, constitute valuable components of the country’s cultural heritage and local economy. These cheeses are the result of long-standing artisanal practices developed in rural and steppe areas to ensure the preservation and valorization of milk under traditional conditions (Benamara et al., 2022; Leksir et al., 2019). 
           
    In the southern and High Plateau regions of Algeria, Jben and Klila are highly appreciated for their nutritional value and their importance in traditional food systems. Jben is a fresh cheese obtained through acid coagulation, while Klila is a dried, shelf-stable product well suited to arid environments due to its reduced moisture content. Both products are produced through natural fermentation processes involving indigenous lactic acid bacteria (LAB), which play a key role in developing their characteristic sensory properties and ensuring product safety (Boumediene et al., 2024; Benamara et al., 2022; Leksir et al., 2019). 
           
    LAB are essential in milk fermentation processes, where they drive acid production, protein breakdown and the synthesis of flavor compounds and exopolysaccharides, while simultaneously limiting the growth of undesirable microorganisms. The microbial composition of these traditional cheeses has been partly characterized: Jben is commonly associated with species such as Lactococcus lactis, Lactiplantibacillus plantarum and Leuconostoc spp., whereas Klila is typically dominated by more acid-resistant bacteria, including Lactiplantibacillus plantarum and Lactobacillus acidophilus. Some of these microorganisms are known to improve product quality and may also contribute to enhanced microbiological safety (Azzouz et al., 2025a; Bendimerad et al., 2024; Benamara et al., 2022; Bouchibane et al., 2022; Ketrouci et al., 2021). 
           
    Although advances have been made in the identification of microbial communities, the functional profiling of indigenous lactic acid bacteria (LAB) from traditional Algerian cheeses remains insufficient, particularly regarding their technological performance and suitability as starter cultures. In particular, comprehensive studies evaluating their acidifying, proteolytic and texturizing capacities in combination are still scarce.
           
    In this context, the present study focused on the isolation and identification of LAB from traditional Jben and Klila cheeses using 16S rRNA gene sequencing. In addition, their technological traits, including acidification ability, proteolytic activity and exopolysaccharide (EPS) production, were investigated. The objective was to select promising strains with potential application as starter cultures, thereby contributing to the preservation and valorization of the unique properties and cultural heritage associated with these traditional dairy products.

    MATERIALS AND METHODS

    Sampling and isolation
     
    This work was conducted at the Laboratory of Sciences and Techniques of Animal Production (LSTPA), in collaboration with the Laboratory of Bioeconomy, Food Safety and Health (LBSAS), Faculty of Natural Sciences and Life, Abdelhamid Ibn Badis University of Mostaganem (Algeria), over the period from January 2024 to December 2025. A total of five samples of traditional cheeses (Jben and Klila), produced from raw cow’s milk, were obtained from artisanal producers in the Djelfa region. LAB isolation was performed by homogenizing the samples in a tryptone-salt solution, followed by cultivation on MRS and M17 agar media at 37°C and 45°C. Colonies exhibiting typical LAB characteristics (Gram-positive and catalase-negative) were selected and subsequently maintained either by periodic subculturing at 4°C or by storage at -20°C in glycerol for long-term preservation (Bouchibane et al., 2023; Bounaama et al., 2022).
     
    Genotypic characterization
     
    Molecular identification of the purified LAB isolates was carried out at the Gene Life Sciences (GLS) Laboratory, University of Sidi Bel-Abbès. Genomic DNA was extracted using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies, Malaysia) and its quality and concentration were assessed using a NanoDropTM One spectro photometer (USA). Amplification of the 16S rRNA gene was performed using the universal primers 27F (5’-AGA GTT TGA TCC TGG CTC AG-32 ) and 1387R (3’-GGG CGG WGT GTA CAA GGC-32 ), following the method described by Lane (1991). PCR reactions were prepared in a final volume of 50 µL, containing 25-50 ng/µL of template DNA, 0.3 µM of each primer, 1.5 µM MgCl‚  and 1.25 U of Hot Start Taq polymerase (Solis Biodyne, Estonia). Amplification was conducted using a SimpliAmp thermocycler (Applied Biosystems, USA). The thermocycling program consisted of an initial denaturation step at 94°C for 12 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min 40 s, with a final extension step at 72°C for 7 min.
           
    PCR amplification products were examined by electrophoresis on 1.5% agarose gels, followed by staining and visualization under ultraviolet illumination. The resulting amplicons were then purified and subjected to bidirectional sequencing using the BigDye Terminator v3.1 kit. After thermal cycling, purification was carried out by ethanol precipitation and sequence fragments were analyzed by capillary electrophoresis using a 3730 XL Genetic Analyzer. The obtained sequences were edited and assembled using CHROMAS PRO software and taxonomic identification was performed through comparison with reference sequences in the NCBI database using the BLAST algorithm. Phylogenetic relationships were subsequently inferred using MEGA12 software based on the Neighbor-Joining method (Edis et al., 2025). 
     
    Technological characterization
     
    pH Kinetics and acidifying activity
     
    Acidifying activity was assessed by measuring pH and titratable acidity in 10% skimmed milk inoculated at 1% and incubated at 30-37°C. Titratable acidity was determined by NaOH titration and expressed in Dornic degrees (°D), calculated (Dahou et al., 2021).
     
    (°D) = VNaOH × 10
     
    Where,
    1/°D corresponds to 0.1/ g of lactic acid per liter of milk.
     
    Proteolytic activity
     
    Proteolytic activity was assessed using PCA medium supplemented with 10% skimmed milk. Sterile Whatman discs inoculated with LAB cultures were placed on the medium and incubated at 30-37°C for 24-72 h. The appearance of a clear zone around the discs indicated proteolytic activity (Bekihal et al., 2025).
     
    Assessment of texturizing ability
     
    This assessment was carried out at the Food Microbiology Laboratory of Istanbul Technical University (Turkey). EPS production was screened on modified BHI agar following anaerobic incubation at 30°C, where viscous and glossy colonies were considered positive. EPS was subsequently extracted and quantified by growing the isolates in modified BHI broth, followed by ethanol precipitation, TCA deproteinization, dialysis (12-14 kDa) and lyophilization (Ispirli et al., 2025). 
     
    Statistical analysis
     
    All experiments were conducted in triplicate (n = 3) and results are presented as mean ± standard deviation. Reproducibility was confirmed by a coefficient of variation below 5%. Data were analyzed using one-way ANOVA with IBM SPSS Statistics 25, followed by Student’s t-test. Statistical significance was set at p<0.05.

    RESULTS AND DISCUSSION

    Sampling and isolation
     
    In this study, ten LAB isolates from two traditional cheeses of the Djelfa province (Jben and Klila) were collected from the localities of Aïn Oussera, Djelfa and Birine. The purified isolates were initially screened for Gram-positive and catalase-negative traits to confirm their identity as LAB (Fig 1).

    Fig 1: Sample of Jben (a), Colony morphology of LAB isolate in MRS (b) and M17 (c), agar medium, Microscopic of LAB isolate (d)


           
    Fig 1 shows the various stages of LAB observation. Colonies grown on MRS and M17 media were circular, smooth and ranged from whitish to cream in color, typical of LAB, indicating their adaptation to mesophilic conditions. Microscopic examination revealed cocci- or rod-shaped cells arranged in clusters or chains, characteristic of LAB commonly present in fermented cheeses. These results confirm the dominance of LAB and demonstrate their morphological diversity, supporting their potential for use in developing starter cultures.
     
    Genotypic characterization
     
    The isolation and identification results presented in Table 1 show that traditional Jben and Klila cheeses from the Djelfa region contain a diverse indigenous LAB community, primarily composed of Enterococcus and lactobacilli from the plantarum/brevis group. Ten isolates were identified through 16S rRNA gene sequencing as members of Enterococcus, Lactiplantibacillus and Levilactobacillus. This combined phenotypic (Fig 1) and molecular approach (Table 1) is commonly employed to characterize indigenous LAB in traditional cheeses and the assignment of accession numbers (PX736099–PX736107, PX737322) ensures traceability and facilitates their future use, as reported by Azzouz et al., (2025b) and Bouchibane et al., (2023).

    Table 1: Source and region of the LAB species identified, including their accession numbers.


           
    The species distribution shows cheese-specific patterns, with E. faecium and L. plantarum dominating in Jben and L. pentosus and Lev. brevis primarily associated with Klila. This pattern aligns with previous studies on traditional Mediterranean and Algerian cheeses, as well as dairy products from the Black Sea region and Morocco. These results confirm that Jben and Klila host characteristic LAB species, underscoring their potential for the development of indigenous starter cultures with both technological and probiotic applications (Azzouz et al., 2025b; Bouchibane et al., 2023).
           
    The phylogenetic tree (Fig 2) displays distinct clustering of isolates by species, showing high similarity to reference strains and confirming their identification via 16S rRNA gene analysis as members of the genera Enterococcus, Lactiplantibacillus and Levilactobacillus. Such concordance between 16S rRNA-based phylogenetic trees and reference taxonomy is widely used to validate bacterial identification, including LAB. The short branch lengths and tight clustering indicate low genetic divergence and strong phylogenetic relatedness, reflecting relatively homogeneous populations (Al-shammary et al., 2025).

    Fig 2: Phylogenetic tree of 16S ribosomal RNA gene partial sequence of LAB species identified, including their accession numbers.


     
    Technological characterization
     
    pH kinetics and acidifying activity
     
    All isolates showed a gradual decrease in pH accompanied by a corresponding increase in titratable acidity (°D) over 24 h, confirming their fermentative activity (Fig 3). Initial measurements were consistent (pH ≈6.70; acidity ≈19°D), reflecting standardized experimental conditions. By 6 h, a moderate acidification phase was observed, with pH values between approximately 5.75 and 6.15 and acidity rising to 24-36°D. After 24 h of incubation, pH values ranged from 4.02 to 5.80, while titratable acidity reached 33-83°D (Table 2).

    Fig 3: Kinetics of pH and titratable acidity of LAB isolates.



    Table 2: Technological activities of LAB Isolated from Jben and Klila.


           
    Statistical analysis revealed highly significant differences among isolates (p<0.01), confirming variability in acidifying performance. Strains IB4 (JDj03) and IB2 (JDj02) showed the highest acidification capacity (pH ≈4.02-4.16; acidity ≈80-83°D), followed by IB7 (JDj04), IB10 (JDj12) and IB5 (LO23), which also exhibited strong acidifying activity (acidity ≥66°D). In contrast, IB3 (JO09) and IB1 (JO01) displayed significantly weaker acidification (p<0.05), with pH values above 5.2 and acidity below 40°D, while IB6 (JB01), IB8 (CDz09) and IB9 (LB10) showed intermediate profiles.
           
    Based on these results, isolates can be classified into three functional groups: strongly acidifying strains (pH drop ≥ 2 units; acidity ≥66°D), moderately acidifying strains and weakly acidifying strains. A clear inverse relationship between pH and acidity was observed, reflecting lactose conversion into lactic acid.
           
    The acidification kinetics observed followed typical lactic fermentation patterns, characterized by a decrease in pH and an increase in titratable acidity within 24 h. Isolates such as JDj03, JDj02 and LO23 exhibited strong acidifying capacity, confirming their suitability as starter cultures, as rapid acidification enhances milk coagulation and microbial safety. Moderately acidifying strains (CDz09, LB10, JB01) may serve as adjunct cultures, while weak acidifiers (JO01, JO09) were more likely involved in flavor development and maturation, as reported by Durango Zuleta et al. (2023) and Sesín et al. (2023).
           
    These findings are supported by the significant pH reduction and acidity values exceeding 60°D observed in active LAB. Overall, the functional diversity among isolates supports the development of multi-strain starter cultures combining complementary properties to improve cheese quality and standardization while preserving traditional characteristics, in agreement with previous studies reported by Grujović et al. (2024), Sesín et al. (2023) and Coelho et al., (2022).
                 
    Proteolytic activity
     
    Proteolytic activity, measured by halo diameter (mm), reflects protein hydrolysis and protease production (Table 2). Halo sizes ranged from 0 to 36 mm, indicating substantial variability among isolates. Statistical analysis showed highly significant differences (p<0.001), confirming pronounced functional heterogeneity. While the high coefficient of variation highlights inter-strain differences, low standard deviations indicate good reproducibility of the measurements. Based on halo diameters, isolates were categorized into three functional groups:
    • Super-proteolytic (≥ 30 mm): JO01 (31 mm), JDj03 (36 mm) and LO23 (36 mm), corresponding to the highest statistical group a (p<0.05).
    • Moderately proteolytic (10-29 mm): JDj02 (20 mm), JB01 (23 mm) and CDz09 (10 mm), falling into intermediate groups b and c.
    • Weakly or non-proteolytic (< 10 mm or 0 mm): JO09, JDj04, LB10 and JDj12, forming a homogeneous group d.
           
    The high activity observed in super-proteolytic strains reflects a strong capacity for casein degradation, promoting peptide formation and flavor development. These strains therefore represent excellent candidates for accelerating cheese ripening and enhancing their bioactive properties, particularly in fresh or ripened cheeses, as reported by Novak et al., (2021). Isolates with moderate activity play an essential intermediate role by ensuring controlled protein hydrolysis. They help limit bitterness and stabilize the rheological properties of the curd, acting as “metabolic bridges” within the microbial ecosystem. In contrast, weakly or non-proteolytic strains were mainly involved in rapid acidification of the medium, contributing to microbiological safety without directly participating in protein degradation, as described by Coelho et al., (2022).
           
    This functional stratification, ranging from highly proteolytic to primarily acidifying strains, reflects a rational approach to starter culture selection. Combining these complementary profiles makes it possible to design optimized mixed cultures capable of synchronizing acidification, proteolysis and flavor development, thereby meeting the technological and sensory requirements of traditional cheeses, according to Coelho et al., (2022) and Novak et al., (2021).
     
    Assessment of texturizing ability
     
    The ability of the ten LAB isolates from Jben and Klila to produce exopolysaccharides was first screened qualitatively on sucrose-enriched modified BHI agar. Only three isolates, JO01 (IB1), LB10 (IB9) and JDj12 (IB10), displayed a clear viscous (“slimy”) phenotype, indicating active EPS synthesis. This observation is consistent with the common use of colony viscosity as a rapid indicator of EPS-producing strains.
           
    Quantitative analysis performed under controlled conditions (30°C, 48 h, anaerobiosis) confirmed these results. After extraction, purification and lyophilization (Fig 4), EPS yields reached 0.69±0.25 g/L for IB1, 0.86±0.30 g/L for IB9 and 0.56±0.20 g/L for IB10 (Table 2), whereas no detectable production was observed for the remaining isolates. The overall production range (0-0.86 g/L) and the relatively high standard deviations highlight pronounced inter-strain variability, which was statistically significant (p<0.05). This heterogeneity reflects the presence of a limited number of efficient producers within a predominantly non-producing population.

    Fig 4: From left to right: bacterial fermentation broth, centrifuged cell pellets, purified EPS after dialysis and finally, lyophilized EPS powder.


           
    The calculated sugar-to-EPS conversion efficiency (based on 7.5 g sucrose/250 mL) ranged from 1.85% to 2.86%, indicating an active but non-optimized metabolic capacity for polysaccharide synthesis. Despite the absence of process optimization, these values suggest that the selected strains possess a favorable baseline for EPS production.
           
    From a technological perspective, EPS-producing isolates are of particular interest due to their ability to improve the rheological properties of fermented dairy products. These polymers contribute to viscosity enhancement, water retention and structural stabilization of the matrix. In contrast, non-producing strains may still play complementary roles, such as acidification and microbial balance, supporting the concept of mixed starter cultures combining EPS-positive and EPS-negative strains to achieve both textural and microbiological benefits.
           
    When compared with literature data, the yields obtained in this study can be considered relatively high for non-optimized systems. For example, the marine isolate MSD8 produced only 0.20 g/L under similar conditions (Abdel-monem et al., 2024), which is markedly lower than the values reported here. This difference suggests that isolates from traditional dairy environments may be naturally better adapted for EPS biosynthesis.
           
    Nevertheless, higher production levels have been described for certain Enterococcus faecium strains. Under rich but non-optimized conditions, strains such as R114 and T52 (isolated from Kishk) reached 2.68 and 2.39 g/L, respectively (Rahnama Vosough et al., 2022), illustrating the strong influence of strain-specific metabolic potential. Furthermore, optimization strategies based on statistical designs (e.g., RSM or CCD) have enabled yields of approximately 2.5-3.2 g/L for strains such as R114, F58 and KT990028 (De Brito et al., 2024; Zanzan et al., 2023; Rahnama Vosough et al., 2021). In some cases, extreme production levels have been achieved, particularly for fish-derived strains such as MC13 and MC-5, reaching up to 11.6 and 16.5 g/L after optimization (Tilwani et al., 2021; Kanmani et al., 2013).
           
    Overall, the EPS yields obtained for IB1, IB9 and IB10 fall within the upper range typically reported for LAB cultivated under non-optimized conditions. Although lower than those achieved after process optimization, these results demonstrate a strong intrinsic production capacity. This baseline performance provides a solid foundation for future improvement through bioprocess optimization and supports the potential application of these strains in food and biotechnological fields.

    CONCLUSION

    The microbial landscape of traditional Djelfa cheeses, Jben and Klila, represents a sophisticated ecological blueprint shaped by centuries of artisanal heritage. Our findings reveal a niche-specific distribution, where Lactiplantibacillus plantarum and Enterococcus faecium drive the fresh profile of Jben, while L. pentosus and Levilactobacillus brevis define the robust character of Klila. This genetic stratification is mirrored by a “technological division of labor”: rapid acidifiers provide microbial safety, while moderate acidifiers, proteolytic and EPS-producing strains orchestrate sensory complexity and the release of health-promoting bioactive peptides. These indigenous consortia do not merely represent local biodiversity; they provide a high-performance toolkit for the design of “terroir-driven” starter cultures. Utilizing these native strains offers a dual advantage: ensuring the industrial reproducibility of traditional textures and flavors while preserving the biological signature of Algerian dairy heritage.

    ACKNOWLEDGEMENT

    The present study was supported by the Faculty of Natural and Life Sciences, Abdelhamid Ibn Badis University. I would like to express my sincere gratitude to the staff of both research laboratories and to the DGRSDT for their valuable support in advancing scientific research in Algeria.
     
    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.
     
    Informed consent
     
    This study does not involve any animal experimentation and no procedures related to the use of animals were conducted.

    CONFLICT OF INTEREST

    The authors declare no conflict of interest.

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