Acidifying activity was evaluated using 100 mL of UHT whole milk with 1 % (v/v) of LAB precultures. The milk cultures were incubated at 37 °C and the evolution of pH (Jaouani et al., 2015) and acidity was monitored after 2, 6 and 24 hours (Jaouani et al., 2015). The pH was measured using a pH meter (Hanna Instruments, Woonsocket, Italy) and the lactic acid was titrated using 0.1 N NaOH (Labkem, Barcelona, Spain) (IDF, 1995).

The protocol of Fguiri et al. (2016) was used to assess the proteolytic activity, with some modifications. The assay was carried out using the well diffusion method where nutrient agar (Biokar, Beauvais, France) was supplemented with 10 % (w/v) skimmed milk (Nestlé, Barcelona, Spain). Fifty microliters of actively growing cells were then inoculated into the wells, and the plates were incubated at 37 °C for 24 hours.

The gas production assay was performed according to Nikita and Develop (2012). Citrate utilization was assessed using the protocol established by Anagnostopoulos et al. (2018) using the Salmonella Typhimurium ATCC 14028 strain as a positive control.

The lipolytic activity was determined according to the protocol of Buffa et al. (2005) with some modifications. The test was carried by well diffusion method using cream fat agar (nutrient agar supplemented with 1 % (v/v) pasteurised milk cream, 38 % fat). Fifty microliters of strains from the exponential phase were then transferred into the wells, and the plates were incubated at 37 °C. The halos’ formation around the wells was measured and documented daily for three days.

The production of pleasant aromas was assessed by inoculating 1 % of isolates from the lag phase into 100 mL of UHT whole milk. The cultures were then incubated at 37 °C for 24 h. The flasks were manually shaken for a few seconds, and the aromas were perceived by smell using the "sniffing assay" (Garabal et al., 2008).

The production of diacetyl-acetoin was screened according to Ribeiro et al. (2013) with a few modifications. Briefly, 0.5 mL of milk-grown strains were added to tubes along with 0.5 mL of 1 % (w/v) α-naphtol (Oxford Lab Fine Chem LLP, Maharashtra, India) and 0.5 mL of 16 % (w/v) KOH (Sigma Aldrich, St Louis, USA). The mixture was further incubated at 30 °C for 10 min. The presence of diacetyl was confirmed by the appearance of a pink ring in the medium.

The antibacterial activity was assessed by the well diffusion method against the following bacteria: Listeria monocytogenes (ATCC19144), S. Typhimurium (ATCC14028), Escherichia coli (ATCC25922) and Staphylococcus aureus (B1). Suspensions of pathogenic bacteria (0.5 MacFarland) were spread over the surface of Muller-Hinton agar plates (Biokar, Beauvais, France). Afterwards, 50 μL of overnight grown LAB cultures were deposited on the wells. The inhibition halos around the wells were measured after 24 hours of incubation at 37 °C (Oldak et al., 2020).

The extraction of genomic DNA was carried out according to Linares-Morales et al. (2020). E. faecalis strains were identified by species-specific PCR with the primers ddlE1 (Forward: 5’-ATC AAG TAC AGT TAG TCT-3’) and ddlE2 (Reverse: 5’-ACG ATT CAA AGC TAA CTG-3’), as described by Dutka-Malen et al. (1995). The reaction mix contained 5 μL of 5× buffer (Bioline, USA), 0.5 μM of primers (Biolegio, Nijmegen, Netherlands), 0.5 μL of Taq polymerase (Bioline, USA), 3 μL of DNA and 7.5 μL of milliQ water. The amplification conditions were as follows: an initial denaturation at 94 °C for 2 min, followed by 35 cycles, which consisted of three steps: denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and an extension step at 72 °C for 1 min. The final extension was carried out at 72 °C for 10 min. The product size was determined using 1 % (w/v) agarose (Bioline, USA) gel, visualised under a Quantum-ST4-3020/WL/20M gel documentation system (Vilber Lourmat, Marne-la-Vallée, France).

The remaining strains (other than E. faecalis) were identified through partial sequencing of the 16S rRNA gene. The primers used for the amplification of the V3 region were V3f (Forward: 5’- CCT ACG GGA GGC AGC AG -3’) and V3r (Reverse: 5’- ATT ACC GCG GCT GCT GG -3’) (Muyzer et al., 1993). The PCR reaction mix was composed of 5 μL of 5× buffer (Bioline, USA), 0.3 μM of primers (Biolegio, Nijmegen, Netherlands), 0.5 μL of Taq polymerase (Bioline, USA), 2 μL of DNA and 16 μL of milliQ water. The amplification conditions were those described by Lorbeg et al. (2014) but with an annealing temperature of 60 °C. PCR products were migrated for 30 min at 100 V in a 1 % (w/v) agarose gel. After the purification steps, the PCR products were sequenced by a 3130xl genetic analyser (Applied Biosystems, USA). The sequences were searched in BLAST of the National Centre of Biotechnology Information (NCBI) database and aligned by the BioEdit software version 7.2.5 (Hall, 1999).

After molecular identification, all E. faecalis strains were excluded, along with some E. faecium strains that did not display antibacterial activity. The remaining 25 isolates were subsequently evaluated for autolysis and safety.

The autolysis percentage was determined according to Bahy El-Din et al. (2002) with minor modifications. Lactococcus and Enterococcus strains were revived in Tryptic-Soy Broth (Biokar, Beauvais, France), while Lb. plantarum and Leuconostoc strains were cultured in MRS broth (Biokar, Beauvais, France). Cultures were centrifuged at 12,000 g for 5 min. Cell pellets were washed twice with (0.01 M, pH=5,5) potassium phosphate buffer (Sigma Aldrich, St Louis, USA) and resuspended in potassium phosphate buffer (0.05 M, pH=6) supplemented with 1 M NaCl. The optical density was adjusted between 0.6 and 0.8 at 650 nm. After the suspensions’ incubation at -20 °C for 24 h and then at 37 °C for another 24 h, the optical density was remeasured. The percentage of autolysis was calculated as follows (Boutrou et al., 1998):

% A = (A0−At)*100A0

Where A₀ is the optical density at t₀ and A_t is the optical density after incubation.

The results were interpreted by genus following the instructions of Ayad et al. (2004).

The haemolytic activity of the selected Enterococcus strains was investigated according to Pino et al. (2019). If clear or green zones appeared around the colonies, the haemolysis was interpreted as β-haemolysis or α- haemolysis, respectively. DNase and gelatinase production were evaluated according to the methods described by Lavilla-Lerma et al. (2013).

The antibiotic susceptibility of the selected strains was tested by the disc diffusion method. In this study, nine different antibiotics were used: erythromycin (15 μg), gentamicin (10 μg), lincomycin (15 μg), norfloxacin (10 μg), penicillin (10 μg), teicoplanin (30 μg), tetracycline (30 μg), trimethoprim-sulfamethoxazole (25 μg), and vancomycin (5 μg). All antibiotics were obtained from Oxoid (Basingstoke, UK).

For the analysis, 200 microliters of LAB precultures (1.5×10⁸ cfu/mL) were spread on the surface of Muller-Hinton agar plates (Biokar, Beauvais, France). Antibiotic discs were placed aseptically with flamed forceps and the plates were incubated at 37 °C. After 24 hours of incubation, inhibition zones were interpreted according to the EUCAST (2021) and CLSI (2020) guidelines.

DNA was extracted using a quick technique described by Espinosa et al. (2013) with slight modifications. Briefly, one colony from a 24 h culture was placed in a PCR tube containing 50 μL of TE buffer (10 mM Tris-HCL, 1 mM EDTA). The tubes were then heated in a thermocycler at 100 °C for 5 min. The lysate was used directly for the PCR reaction.

The amplification of vanA and vanB genes was conducted using the following primer pairs: A1 (Forward: 5'-GGGAAAACGACAATTGC-3') and A2 (Reverse: 5'-GTACAATGCGGCCGTTA-3'), resulting in a 732 bp product size for the vanA gene; and B1 (Forward: 5'-ATGGGAAGCCGATAGTC-3') and B2 (Reverse: 5'-GATTTCGTTCCTCGACC-3'),yielding a 635 bp product size for vanB gene (Dutka-Malen et al., 1995). The PCR reactions were performed in a 25 μL reaction mixture containing 5 μL of buffer (Bioline, USA), 0.6 μM of primers (Biolegio, Nijmegen, Netherlands), 0.5 μL of dNTPs mix (Promega, Madison, USA), 0.5 μL of Taq polymerase (Bioline, USA) and 2 μL of DNA template. The PCR mixtures were subjected to the amplification conditions described by Asadpour and Ghazanfari (2019).

The identification of LAB isolates by specific PCR and partial 16S rRNA sequencing is presented in Table 1. The acidifying activity of the different LAB groups after 6 and 24 hours of incubation is presented in Table 2A.

In this study, 67.4 % of the isolates successfully reduced the milk's pH from 6.6 to 5.3 within 6 hours, and 71.9 % lowered the pH to below 4.8 after 24 h, meeting the criterion established by Cachon et al. (2002) for good acidifiers (Table 2B). Interestingly, despite being members of nonstarter lactic acid bacteria (NSLAB), Lb. plantarum strains exhibited the highest pH reductions (ΔpH 2.7-2.8) after 24 hours of incubation. In particular, the strains 18.1 and 18.6 achieved a ΔpH of 2.8, with Dornic acidity levels of 120.2 °D and 117.9 °D, respectively (Table 3). These values were comparable to those of Lb. plantarum strains isolated from Serpa PDO cheese (Araújo-Rodrigues et al., 2021) and were significantly higher than the results reported by Fguiri et al. (2016). Enterococcus isolates also demonstrated good acidifying properties by lowering the pH below 4.8 after 24 hours (Table 2A), aligning with the findings of Albayrak and Duran (2021).

Table 1. Isolates identification by specific PCR and partial 16S sequencing

Table 2. A. Acidifying activity of LAB strains isolated from Moroccan Jben cheese

The mean values presented in the table are calculated from two replicates for each sample.

B. Percentage of isolated strains meeting desired thresholds in acidifying activity

Table 3. LAB strains isolated from Jben cheese exhibiting notable technological properties

Significant differences (p<0.05) were observed among strains for pH at 6 hours, pH at 24 hours, lactic acid production at 24 hours, and proteolytic activity. Means with different superscripts in the same column indicate significant difference. LA= Lactic acid; -= no production; +=low production; ++ =moderate production; +++=high production; ++++ =very high production

The acidifying ability of the strains was also categorized based on pH reduction units after 6 hours of incubation resulting in four groups: "fast acid producers," which lowered the pH by more than 2 units; "medium acid producers," showing a decrease of 1.5 to 1.9 pH units; and "slow acid producers", resulting in a pH decline of 1 to 1.4 pH units. The largest group was that of "medium acid producers" comprising 52.8 % of the strains, and the smallest group was that of "fast acid producers" with 4.5 % of the strains. Regarding Lactococcus species, only 5.9 % of the isolates were classified as fast acidifiers, while 58.8 % were categorized as medium acidifiers. However, it is noteworthy that all Lactococcus strains, including the slow acidifiers, were capable of producing curd after 24 hours of incubation. The unique Ln. mesenteroides 17.5 strain, however, was classified as a slow acid producer and failed to produce a coagulum from whole milk, which is consistent with the results of Zarour et al. (2018).

Proteolysis enhances both the flavor and texture of cheese, with LAB - particularly NSLAB - being the primary organisms responsible for this activity (Hutkins, 2006). In our study, all strains with acidifying properties also exhibited proteolytic activity (Table 4A). According to Vuillemard et al. (1986), strains are considered suitable for cheesemaking if they produce proteolysis halos of 15-21 mm. In this study, 6.74 % of the isolates demonstrated proteolytic activity within this range (Table 4B), with Lb. plantarum isolates showing the largest proteolytic halos, surpassing those of other genera. Regarding Enterococcus isolates, most demonstrated low to moderate proteolytic activity, in line with the findings of Pisano et al. (2019). Similarly, the Lactococcus genus was classified as moderately proteolytic, while the Leuconostoc genus exhibited weak proteolytic activity, consistent with the results of Zarour et al. (2018).

Table 4. A. Proteolytic activity of LAB strains isolated from Moroccan Jben cheese

The mean values presented in the table are calculated from two replicates for each sample.

B. Percentage of isolated strains meeting desired thresholds in proteolytic activity

*Halo

Lactic acid bacteria are generally considered to be weakly lipolytic. However, they can release small amounts of fatty acids in cheeses during extended ripening. Indeed, several lipases/esterases originating from strains of Lactococcus and Lactobacillus have been isolated and characterised (McSweeney, 2011). In the present study, none of the isolates exhibited lipolytic activity, consistent with findings by Câmara et al. (2019) and Yerlikaya (2019), who observed no lipolysis haloes in species from the Enterococcus, Lactococcus, Lactobacillus, and Leuconostoc genera when incubated in tributyrin agar.

Present in milk at low concentrations, citrate is metabolised by limited LAB species, primarily belonging to the genera Leuconostoc, Lactococcus, and Lactobacillus. This metabolic process not only yields CO₂, crucial for the formation of holes in certain cheeses, but also produces diacetyl and acetate, essential aromatic compounds found in soft cheeses and fermented milks (Parente et al., 2017). However, in the present study, none of the strains was able to use citrate, which is consistent with the results obtained by Anagnostopoulos et al. (2018).

Based on the gas production assay, all strains were homofermentative except for Ln. mesenteroides 17.5, which produced gas bubbles after incubation (Table 3). This observation aligns with established knowledge, as genera known for heterofermentative activity include Leuconostocs, Oenococci, Weissellas, and certain Lactobacilli (Axelsson, 2004).

Diacetyl is one of the most important volatile compounds found in cheese (Broadbent et al., 2011). Beyond its sensory attributes, diacetyl is also recognized for its antimicrobial properties against yeasts, Gram-negative, and Gram-positive bacteria (Molloy et al., 2011). In this study, all Lb. plantarum strains and the majority of E. faecalis (91.2 %) and E. faecium (90.9 %) isolates exhibited diacetyl production. This finding aligns with previous studies indicating that Enterococcus and Lactobacillus species are the primary diacetyl producers among lactic acid bacteria (Domingos-Lopes et al., 2017). Certain species of the genus Leuconostoc, such as Ln. mesenteroides subsp. cremoris and Lc. lactis cit+, are also known diacetyl producers (Marsili, 2011). However, our analysis of the isolate Ln. mesenteroides 17.5 (Table 3) revealed no diacetyl production, corroborating the findings of Saidi et al. (2020). Regarding the Lactococcus isolates, diacetyl production was observed in 47.1 % of strains. Although this percentage is lower compared to the Lb. plantarum and Enterococcus isolates, in this study, it is still higher than that reported by Perin et al. (2017), who observed diacetyl production in only one out of 23 Lactococcus strains.

Notably, although citric acid is a well-established precursor of diacetyl, our isolates were found to produce it through alternative metabolic pathways, as none could use citrate as their only carbon source. These findings are consistent with those reported by Saidi et al. (2020). These pathways likely involve pyruvate derived from sugar metabolism or the acetaldehyde route, bypassing direct citrate utilization (Escamilla-Hurtado et al., 1996).

Before using lactic acid bacteria as starters or additives in cheese, their ability to produce pleasant flavours must be evaluated. In this study, 62.7 % of the isolates have been found to emit pleasing aromas, mostly reminiscent of the fresh smell of yoghurt and butter (Tables 3, 5 and 6). The majority of E. faecalis and E. faecium strains produced pleasant flavours, consistent with previous reports on the ability of Enterococcus strains to produce flavouring compounds in milk (Câmara et al., 2019). In the Lactococcus group, over half of the strains (52.9 %) produced sulphur aromas, a prevalent trait among Lc. lactis strains, as confirmed by the findings of Garabal et al. (2008). The 47.1 % remaining Lactococcus isolates produced a discernible yoghurt aroma. Interestingly, a noticeable yoghurt-vanilla flavour was detected in two Lc. lactis distinct strains: 5.12 and 12.1 (Table 3). The Ln. mesenteroides 17.5 strain produced a cooked milk flavor (Table 3), a characteristic previously observed in nearly half of the Leuconostoc isolates reported by Garabal et al. (2008). This result may be due to the weak acidifying and proteolytic activities of Leuconostoc species, which likely limit their ability to produce desirable aromas.

Table 5. Aroma production by LAB strains isolated from Moroccan Jben cheese

*Indicates that no strain produced this aroma

The assessment of the antibacterial activity against four different pathogens - L. monocytogenes (ATCC19144), S. Typhimurium (ATCC14028), E. coli (ATCC25922), and Staph. aureus (B1) -revealed significant differences (p<0.05). Lactic acid bacteria produce various bioactive compounds with antimicrobial properties, including lactic acid, diacetyl, ethanol, fatty acids, and bacteriocins such as nisin (Molloy et al., 2011). In our study, noteworthy percentages of E. faecalis (70.6 %) and E. faecium (27.2 %) strains inhibited the growth of L. monocytogenes (ATCC19144) (Figure 1), aligning with the findings reported by Albayrak and Duran (2021). Among these, E. faecium 15.2 showed the largest inhibition zone diameter of 15 mm, followed closely by E. faecalis strains 9.3 and 1.13, which both exhibited diameters of 14 mm (Figure 1). However, isolates from Lactococcus, Lactiplantibacillus, and Leuconostoc genera showed no antibacterial activity.

In this study, four virulence determinants - haemolysis, gelatinase activity, DNase activity, and antibiotic resistance - were evaluated in E. faecium strains (Table 7). None of the isolates showed β-haemolysis, gelatinase or DNase activity. However, the strains 15.2 and 13.7 showed partial haemolysis (α- haemolysis) and were then excluded from the collection.

Table 7. Safety aspects of selected E. faecium strains isolated from Moroccan Jben cheese

*ND - not determined