Antimicrobial activity of Lactobacillus spp. isolated from fermented foods and their inhibitory effect against foodborne pathogens

Sampling and isolation of Lactobacillus spp. from fermented foods

Fermented food samples including cheese, pickles and yoghurt were collected from various markets in April 2021. Samples were transported under sterile conditions and stored at 4 °C. Each sample (1 g) was dissolved in 9 mL of MRS broth and blended using a Stomacher machine (Stomacher 400, Seward, West Sussex, UK) for 5 min. The inoculated tubes were incubated at 37 °C for 48 h under anaerobic conditions. Tenfold serial dilutions were conducted, and subculturing was performed on solid MRS medium. After incubation, prominent white colonies were purified on MRS agar plates. Identification involved Gram staining, selective agar growth and biochemical tests. Acid production ability was tested on MRS. Only Gram positive, catalase, and oxidase-negative isolates were considered potential Lactobacillus spp. candidates. Stock cultures were kept at −20 °C in 15% (v/v) glycerol. Molecular identification of all isolated strains from each sample was carried out following the procedure of Wang et al. (2020). Genomic DNA was extracted from all bacterial isolates using a DNA extraction kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. The quantity of extracted DNA was measured using a Nanodrop One spectrophotometer (Thermo Scientific, Waltham, MA, USA). This DNA was used as the template for PCR amplification of the 16S rRNA gene sequences from each strain. The primers for amplification, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′, that amplified a region of approximately 1,500 bp. The PCR reaction used 25 µL of OneTaq Quick-Load 2X Master Mix with Standard Buffer (New England BioLabs, Ipswich, MA, USA), 1 µL of forward primer at 10 pmols/µL, 1 µL of reverse primer at 10 pmols/ µL, 1.5 µL of DNA template, and 9 µL of nuclease-free water. PCR was performed using a (Biometra TPersonal thermal cycler, Analytik, Jena, Germany) under the following conditions: initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min. The resulting PCR products were confirmed by agarose gel electrophoresis, purified and sequenced. Sequence identity was determined using BLAST (Nucleotide BLAST, 16S rRNA sequence database). Electrophoresis was performed at 80 volts for 45 h on a 1.5% agarose gel stained with FloroSafe DNA Stain (1 µL/mL 1st BASE, Singapore). The following strains were identified from various fermented food samples: Lactiplantibacillus plantarum from yoghurt, Lactobacillus paracasei from feta cheese, Lactiplantibacillus plantarum from kafir cheese, Lactiplantibacillus plantarum from Syrian cheese, Lactobacillus paracasei from Turkish cheese, Lactiplantibacillus pentosus from mixed pickles, and Lactobacillus paracasei from green olive pickles.

Screening of Lactobacillus spp. for antagonistic activity

Detection of bacteriocin synthesis genes in Lactobacillus spp.

The presence of 10 bacteriocin-related genes was investigated in seven Lactobacillus isolates by PCR using specific primers. The PCR primers and annealing temperatures are presented in Table 1. PCR conditions were similar for all genes: an initial denaturation step at 94 °C for 5 min, followed by 34 cycles of denaturation at 94 °C for 30 s, an extension at 68 °C for 60 s, and a final extension at 68 °C for 10 min (Macwanaa & Muriana, 2012). The PCR products were identified by electrophoresis on 1.5% agarose gels stained with FloroSafe DNA Stain.

Effects of temperature, pH, and enzymes on L. plantarum KR-3 CFS

To analyse pH tolerance, L. plantarum KR-3 CFS was adjusted to pH 2.0, 4.0, 6.0, 8.0, and 10.0 by using HCl (3 mol/L) or NaOH (3 mol/L). After 1 h at 37 °C, samples were readjusted to their initial pH. For thermal stability, L. plantarum KR-3 CFS was exposed to 25 °C, 40 °C, 60 °C, 80 °C, and 100 °C for 1 h. Enzyme treatments involved adding proteinase K, pepsin, and lysozyme (1 mg/mL) into the CFS, incubating at 37 °C for 2 h, and inactivating at 80 °C for 10 min. Untreated CFS served as the control. Antibacterial efficacy against bacterial pathogens was assessed using the Oxford cup double-layer plate method. This experiment was repeated three times (Hwanhlem, Chobert & H-Kittikun, 2014).

Biofilm eradication concentration determination method

The efficacy of L. plantarum KR3 CFS against biofilm formation was assessed in 96-well plates (Nest Biotechnology, Wuxi, China) following the method outlined by Jiang et al. (2022). L. plantaraum KR-3CFS was diluted to concentrations of 1 × MIC and 2 × MIC, and cultures of S. aureus, E. coli, and Salmonella spp. at a concentration of (10⁷ CFU/mL) were added to the microplates containing various dilutions of CFS. Each well of the 96-well microtiter plate was filled with 100 µL of bacterial suspension and 100 µL of CFS, resulting the total volume in each well to 200 µ. Negative control wells were filled with 200 µL of media only. After mixing, the microplates were incubated for 24 h at 37 °C, the supernatant was removed, and the residue was washed with PBS. The biofilm biomass was stained with 0.1% crystal violet solution. Following a 15 min incubation period at room temperature, the plates were washed and allowed to air dry, followed by another washing step to remove excess dye. The retained crystal violet, indicating biofilm biomass, was solubilized with acetic acid and the absorbance was measured at 595 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, USA). The experiment was performed in triplicate.

Scanning electron microscopy analysis

Approximately 3 mL of S. aureus, E. coli, and Salmonella spp. culture, each with a concentration of 10⁷ CFU/mL was centrifuged at 7,104× g at 4 °C for 3 min. Afterward, the harvested cells were washed once with PBS buffer and then resuspended in 1 mL of L. plantaruum KR3 at 37 °C for 2 h. The treated bacterial cells were fixed with 2.0% glutaraldehyde at 4 °C for 8 h and dehydrated in a series of ethanol solutions with increased concentration (30% to 100%) for 15 min each. The samples were then transferred onto polished silicon wafers measuring 10 × 10 mm² and left to dry at 25 °C. After drying, the samples were coated with gold powder before imaging under a scanning electron microscope (JEOLJSM 6510 IV SEM). Untreated S. aureus, E. coli, and Salmonella spp. cells served as the control. Each experimental condition was performed in triplicate (Li et al., 2015).

Statistical analysis

Data analysis was performed, and results are presented as the mean ± standard deviation of three independent replicates. One-way ANOVA was conducted to assess significance levels among multiple groups. A P value < 0.01 was considered significant.

Detection of antimicrobial activity

The antibacterial activity of Lactobacillus strain was evaluated using two methods the well-diffusion assay and the double-layer agar-overlay method. To eliminate the influence of acid production, the supernatant from each isolate was neutralized with 1M NaOH before testing. A total of seven isolates were screened for antimicrobial properties against both Gram-negative and Gram-positive bacteria (Fig. 2). The well-diffusion and double-layer assays measured the diffusion of antimicrobial compounds produced by Lactobacillus spp. through the agar medium, as described by Strus (1998). The size of the inhibition zones depended on the sensitivity of the target strain and the assay method employed. Across both methods, not all Lactobacillus strains exhibited inhibitory effects against both Gram-negative and Gram-positive bacteria.

In terms of effectiveness, the double-layer agar-overlay method generally produced larger inhibition zones compared to the well-diffusion assay. For example, the highest inhibition zone for Lb3 against Salmonella spp. was 22.0 mm using the double-layer method, while the well-diffusion assay produced a smaller inhibition zone of 14.0 mm. This suggests that the double-layer method is more sensitive in detecting antimicrobial activity.

Furthermore, the well-diffusion assay showed greater variability in inhibition zone sizes among different isolates, whereas the double-layer method provided more consistent results across all strains. The most significant inhibition zone observed across both methods was 22.0 mm, recorded using the double-layer method.

These findings indicate that while both methods are effective, the double-layer agar-overlay method proved to be more reliable for assessing the antagonistic effects of crude antimicrobial compounds from Lactobacillus strains against S. aureus, E. coli, and Salmonella spp. (Fig. 2).

Gene screening for bacteriocin production

Table 4 illustrate the genetic variability of bacteriocin genes across different Lactobacillus strains and L. plantarum, providing insights into their bacteriocin production capabilities. In this study, seven strains of Lactobacillus were examined for the presence of genes associated with the pln locus, known to be involved in bacteriocin production.

The screening revealed notable genetic diversity among the strains. The genes plnA, plnB, plnEF, plnL, and plnNc8IF were consistently detected across all isolates, indicating their fundamental role in bacteriocin production. Among the identified genes, plnA and plnB were the most frequently found, followed by plnD and plnC (Diep et al., 2003; Atieh et al., 2021).

This genetic variability suggests differences in the bacteriocin production capabilities of various Lactobacillus strains, which could be harnessed for different biotechnological applications. The consistent presence of key genes like plnEF and plnL in all strains highlights their importance in bacteriocin synthesis, potentially contributing to the strains’ ability to inhibit pathogenic bacteria and withstand gastrointestinal conditions.

Numerous studies have focused on the bacteriocin production of Lactobacillus strains, particularly their ability to inhibit pathogens. Our findings further emphasize the technological relevance of these strains, especially those sourced from fermented foods, and their potential applications in industrial processes due to their probiotic and functional properties.

Determination antibacterial activity of L. plantarum KR3

The antibacterial activity of L. plantarum KR3 CFS was evaluated against S. aureus, E. coli and Salmonella spp. Compared with the control group (MRS medium), CFS derived from L. plantarum KR3 exhibited substantial inhibitory effects against all tested pathogens. The inhibition-zone diameters were measured at 20.0 ± 0.34 mm against S. aureus, 23.0 ± 1.64 mm against E. coli, and 17.1 ± 1.70 mm against Salmonella spp. (all P < 0.01), indicating significant antibacterial activity (Fig. 3).

Due to its pronounced antibacterial activity, L. plantarum KR3 CFS was selected for further analysis because of its significant antibacterial activity against a range of pathogens, including S. aureus, E. coli, and Salmonella spp. It produced the largest inhibition zones in preliminary assays, indicating a strong potential for bacteriocin production. Furthermore, L.plantarum KR3 consistent effectiveness highlights its potential as a reliable natural antimicrobial agent, making it a promising candidate for applications in food safety and preservation.

Effects of temperature, pH, and enzymes on L. plantarum KR3 CFS

The stability of L. plantarum KR3 CFS against variations in pH, heat, and enzymatic treatments is depicted in Fig. 4. Across pH 2, 4, and 6, no significant decrease in antibacterial activity was observed (P > 0.05). However, notable reductions in activity were evident at pH 8 and 10 compared with the control (P < 0.05), although considerable activity was retained with 74.8% ± 0.71% and 63.4% ± 0.60% preserved, respectively.

The antimicrobial effectiveness of L. plantarum KR3 CFS remained relatively stable across pH levels ranging from 2 to 6. Even at pH 8 and 10, a substantial proportion of its activity was preserved, with 74.8% ± 0.71% and 63.4% ± 0.60% retained, respectively (Fig. 4A).

Moreover, the thermal stability of L. plantarum KR3 CFS was exceptional, with 95.3% of its antibacterial activity retained even after exposure to 100 °C for 60 min (Fig. 4B). Furthermore, no significant reduction in antibacterial effectiveness was observed when subjecting L. plantarum KR3 CFS to temperatures ranging from 40 °C to 100 °C (P > 0.05).

Subsequent enzymatic treatments revealed a notable decrease in the antimicrobial potency of L. plantarum KR3 CFS, with reductions of 24.0% ± 0.55% and 54.1% ± 0.55% observed after treatment with proteinase K and pepsin, respectively (P < 0.05 compared with the untreated control). Conversely, exposure to lysozyme did not result in a significant alteration in antibacterial activity (P > 0.05 compared with the untreated control) (Fig. 4C).

Biofilm eradication concentration determination method

The cell free supernatant (CFS) of L. plantarum KR3 exhibited significant anti-biofilm activity against common foodborne pathogens, consistent with findings from previous studies and expanding our understanding of probiotic interventions in food safety. The observed anti-biofilm percentages were 59.12 ± 0.03% against S. aureus, 60.03 ± 0.04% against Salmonella spp.and 83.50 ± 0.01% against E. coli, respectively (P < 0.05 for S. aureus and Salmonella spp., P < 0.01 for E coli.) compared with untreated controls). Furthermore, at 1x MIC, L. plantarum KR3 demonstrated even higher anti-biofilm activity against Salmonella spp. and E. coli, surpassing previous findings. As well, a clear picture of the damage in the food pathogens cells following cell free supernatant (CFS) compounds bacteriocins treatment was observed through SEM. The electron micrograms shown in Fig. 5 indicate that bac the cell-free supernatant (CFS) compounds exhibits an antibiofilms effect against the cells.

Detection of antimicrobial activity

The antibacterial activity of lactic acid bacteria (LAB), assessed using well diffusion and double-layer assay methods, reveals key insights into their potential as natural antimicrobial agents. The study demonstrated that while not all LAB isolates exhibited inhibitory effects against both Gram-negative and Gram-positive bacteria simultaneously, some strains showed significant antimicrobial properties. This variation can be attributed to the differing sensitivities of target strains to the antimicrobial compounds produced by Lactobacillus spp., as well as the detection methods used.

The highest inhibition zone observed was 22 mm, highlighting the effectiveness of the cell free supernatant (CFS) compounds produced by certain Lactobacillus isolates, particularly against common foodborne pathogens such as S. aureus, E. coli, and Salmonella spp. This finding is consistent with prior research, including that of Strus (1998), who emphasized the importance of detection methods in evaluating the antibacterial activity of LAB.

In comparing these results with other studies, Yerlikaya, Saygılı& Akpınar (2020) found that lactic acid bacteria isolated from Burkinabe fermented milk products also exhibited significant antimicrobial activity. The study by Yazgan et al. (2020) demonstrated that LAB can produce bacteriocins, organic acids, and hydrogen peroxide, which collectively contribute to their antibacterial properties. This aligns with our findings that neutralized supernatants of LAB still retain considerable antimicrobial activity, indicating the presence of active compounds in the CFS as the primary inhibitory compounds.

Furthermore, the modified agar overlays method proved to be the most reliable for determining the antagonistic effect of these active compounds in the CFS. This is supported by Peng et al. (2021), who also found that well diffusion assays may underestimate the antibacterial activity due to the diffusion limitations of larger molecules like bacteriocins. In terms of practical applications, the ability of Lactobacillus spp. to produce effective bacteriocins suggests their potential use in food preservation and safety.

Gene screening for bacteriocin production

The genetic variability in bacteriocin production among Lactobacillus strains, as detailed in Table 2, underscores the diversity and potential of these bacteria in biotechnological applications. The presence or absence of specific bacteriocin genes such as plnA, plnB, plnEF, plnL, and plnNc8IF across different isolates of L. plantarum and L. paracasei highlights their fundamental roles in bacteriocin production.

Our findings align with existing literature, which emphasizes the genetic diversity in bacteriocin-producing Lactobacillus strains. For instance, Maldonado, Ruiz-Barba & Jiménez-Díaz (2003) identified plnA, plnB, and other pln genes in L. plantarum NC8, which are crucial for bacteriocin production. Similarly, Ben Omar et al. (2008) highlighted the prevalence of plnEF and other pln genes in various Lactobacillus strains, indicating their conserved nature across different species.

Comparative studies also support the technological relevance of these genes. For example, Rizzello et al. (2014) investigated the presence of plnG and plnH genes, involved in the ABC transport system, in several Lactobacillus strains, demonstrating their role in bacteriocin transport and immunity. This genetic diversity is not just an academic interest; it has practical implications for the use of Lactobacillus strains in food preservation and probiotic applications.

The variability in bacteriocin gene presence also indicates the adaptability of Lactobacillus strains to different environments and their potential to inhibit a wide range of pathogens. This is particularly relevant in the context of food safety, where the ability to inhibit pathogenic bacteria is crucial. Ouoba (2017) demonstrated that Lactobacillus strains from fermented foods exhibit significant antimicrobial activity, which is partly attributed to the production of bacteriocins.

Furthermore, our study’s focus on genes associated with the pln locus is supported by the work of researchers like Strus (1998), who emphasized the importance of these genes in determining the antimicrobial properties of Lactobacillus spp. These results align with previous research showing the widespread occurrence of the pln locus in Lactobacillus spp., which plays a crucial role in their antimicrobial properties (Sáenz et al., 2009; Kawahara, Takeshi & Hiromi, 2022). In our study, we consistently observed the presence of the plnA, plnB, plnEF, and plnL genes, which are key contributors to the antimicrobial activity.

Determination antibacterial activity of L. plantarum KR3

The antibacterial activity of L. plantarum KR3 CFS against S. aureus, E. coli, and Salmonella spp. demonstrates the significant potential of this strain as a natural antimicrobial agent. Compared with the control group (MRS medium), the CFS derived from L. plantarum KR3 exhibited substantial inhibitory effects against all tested pathogens, with inhibition-zone diameters of 20.0 ± 0.34 mm against S. aureus, 23.0 ± 1.64 mm against E. coli, and 17.1 ± 1.70 mm against Salmonella spp. (all P < 0.01). These findings indicate significant antibacterial activity and align well with previous studies highlighting the efficacy of Lactobacillus strains in pathogen inhibition.

Comparative studies provide further context to these results. For instance, Wayah & Philip (2018) and Peng et al. (2021) found that L. plantarum strains possess strong antimicrobial properties against a range of pathogens, consistent with our findings. Their study reported similar inhibition zones, demonstrating that the production of bacteriocins, organic acids, and hydrogen peroxide by L. plantarum strains plays a critical role in their antibacterial activity. Additionally, Qiao et al. (2021) emphasized the importance of these antimicrobial compounds in inhibiting foodborne pathogens, supporting the observed results of our study.

Effects of temperature, pH, and enzymes on L. plantarum KR-3 CFS

The study showed that the antibacterial activity of L. plantarum KR3 CFS remained relatively stable across pH levels ranging from 2 to 6, with no significant decrease in activity observed (P > 0.05). However, notable reductions in activity were evident at pH 8 and 10 (P < 0.05), although considerable activity was still retained with 74.8% ± 0.71% and 63.4% ± 0.60% preserved, respectively. This aligns with findings by Arrioja-Bretón et al. (2020), who demonstrated that Lactobacillus strains maintain inhibitory activity at lower pH levels, likely due to the stability of organic acids produced by these bacteria, which are effective antimicrobial agents at acidic pH.

The thermal stability of L. plantarum KR3 CFS was exceptional, with 95.3% of its antibacterial activity retained even after exposure to 100 °C for 60 min. Additionally, no significant reduction in antibacterial effectiveness was observed when subjecting the CFS to temperatures ranging from 40 °C to 100 °C (P > 0.05). This is consistent with previous findings by Crowley, Mahony & Van Sinderen (2013), who highlighted the heat resistance of organic acids produced by Lactobacillus strains. This high level of thermal stability makes L. plantarum KR3 CFS a promising candidate for use in heat-treated food products.

Enzymatic treatments revealed a notable decrease in the antimicrobial potency of L. plantarum KR3 CFS, with reductions of 24.0% ± 0.55% and 54.1% ± 0.55% observed after treatment with proteinase K and pepsin, respectively (P < 0.05 compared with the untreated control). This suggests that proteinaceous compounds, possibly bacteriocins, contribute significantly to the overall antimicrobial activity of the CFS. Conversely, exposure to lysozyme did not result in a significant alteration in antibacterial activity (P > 0.05 compared with the untreated control). This differential impact of enzymatic treatments underscores the complexity of the antimicrobial mechanisms at play, likely involving a combination of bacteriocins and organic acids. These findings are consistent with those of Ju et al. (2021), who reported that the bacteriocins produced by Lactobacillus strains exhibit strong antimicrobial properties and are relatively stable under various conditions. The stability of these compounds across a range of environmental factors supports their potential application in diverse industrial processes, particularly in food preservation.

Biofilm eradication concentration determination method

Lactobacillus isolate number three, identified as L. plantarum KR3, was selected from the seven isolates for its significant antibacterial activity against common foodborne pathogens demonstrating strong applicability in the context of food safety. The observed anti-biofilm percentages were 59.12 ± 0.03% against S. aureus, 60.03 ± 0.04% against Salmonella spp. and 83.50 ± 0.01% against E. coli. This aligns with and expands upon previous studies, further illustrating the effectiveness of probiotic interventions in controlling biofilm formation.

These indicator strains form biofilms as a survival mechanism due to their ability to produce extracellular polymeric substances (EPS), which facilitate adhesion to surfaces and protect against environmental stresses. Factors such as temperature, pH, and nutrient availability promote biofilm formation, enhancing their survival and resistance to antimicrobial agents. For instance, S. aureus produces polysaccharide intercellular adhesin (PIA) to adhere to surfaces (Nasser et al., 2022), while E. coli utilizes fimbriae and pili for attachment (Rossi, Paroni & Landini, 2018). Similarly, Salmonella spp.uses its flagella for motility and surface attachment, contributing to its biofilm development (Maruzani et al., 2019).

The high anti-biofilm activity against E. coli and Salmonella spp. at 1MIC is consistent with studies by Monteagudo-Mera, Rastall & Gibson (2019) or even surpasses the results of earlier research, indicating an enhanced effectiveness of L. plantarum KR3. This is corroborated by the study of Peng et al. (2021), which also highlighted the robust antimicrobial properties of L. plantarum strains. The significant inhibition of biofilm formation by these pathogens suggests a strong potential for L. plantarum KR3 in mitigating biofilm-associated risks in food processing environments.

The use of scanning electron microscopy (SEM) provided visual evidence of the damage inflicted on the food pathogen cells by compounds in the CFS. This reinforces the understanding that these compounds play a crucial role in disrupting biofilm structures, a finding consistent with research by Moridi et al. (2020). Their study emphasized the diverse range of antimicrobial compounds found in Lactobacillus CFS, such as hydrogen peroxide, oxygen metabolites, exopolysaccharides, and saturated fatty acids, which act as biosurfactants and contribute to anti-biofilm properties (Lagrafeuille et al., 2018).

Similar studies have demonstrated the efficacy of Lactobacillus strains in biofilm eradication. For instance, Ju et al. (2021) reported the effective biofilm removal capabilities of Lactobacillus strains against pathogens like S. aureus. The consistency of these results across different studies underscores the reliability of Lactobacillus strains, particularly L. plantarum KR3, in combating biofilm formation and enhancing food safety protocols. The findings from this study highlight the promising application of L. plantarum KR3 CFS as a natural antimicrobial agent in food preservation and safety. By effectively inhibiting biofilm formation, these probiotics can reduce the risks posed by biofilm-associated pathogens in food processing and storage environments. The use of such natural interventions aligns with consumer preferences for clean-label products and offers an alternative to chemical preservatives.