Isolation and Optimization of Bacteriocin-Producing Lactic Acid Bacteria from Nigerian Fermented Foods

2.1 Isolation of lactic acid bacteria from Nigerian fermented foods

The fermented foods used in this study included ogi, fufu, and iru, which are widely consumed traditional fermented foods in Nigeria. Ogi is a fermented cereal slurry produced mainly from maize, sorghum, or millet and commonly consumed as breakfast porridge. Fufu is a fermented cassava product prepared through soaking, fermentation, and pounding of cassava tubers. Iru is a fermented condiment produced from African locust beans (Parkia biglobosa), commonly used as a flavouring agent in soups. These fermented foods are rich sources of diverse lactic acid bacteria that contribute to fermentation and food preservation.

Isolation of LAB isolates was performed using de Man Rogosa Sharpe Agar (MRS) supplemented with 0.05 g fluconazole. Fifteen grams of each food sample was dissolved in 150 mL sterile normal saline (0.85 w/v NaCl) and serially diluted 10-fold in sterile distilled water. LAB were isolated on a standard pour plate. Fifteen milliliters of sterile MRS Agar, in a known dilution in a 1 mL sterile Petri dish, was precooled at 45°C. The plates were left to dry and were placed in an incubator at 35°C ± 2°C for 48 h. Colonies of various morphologies were randomly selected after incubation with a flamed platinum wire loop, streak plated and subcultured on MRS agar plates to obtain pure colonies. The respective pure colonies were kept as slants in bottles with sterile MRS agar and kept in a refrigerator (4°C ± 2°C) until required.

2.2 Preliminary antibacterial screening

The indicator pathogens (Bacillus subtilis, Bacillus proteolyticus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Pseudomonas fuscoginae) were cultured in nutrient broth and incubated at 37°C for 18–24 h prior to use. The cultures were adjusted to approximately 10⁶ CFU/mL before inoculation. Preliminary antibacterial screening was carried out using (48 h old broth) cultures of the LAB strains against selected pathogens (Salmonella typhi, Bacillus subtilis, Bacillus proteolyticus, Xanthomonas Campestris, Pseudomonas aeroginosa, Klebsiella pneumonia, and Pseudomonas fuscoginae) using the agar-well diffusion method as reported by Akpor et al. (2022) and Reuben et al. (2019). To 200 mL sterile molten nutrient agar in a 250 mL capacity Erlenmeyer flask, 1 mL of 24 h old broth culture of the respective test pathogen was inoculated and swirled gently and mixed thoroughly several times. Following mixing, 20 mL of cooled molten agar with the inoculated pathogen was dispensed in sterile petri dishes and left to solidify. Following solidification, four holes were bored on each plate, using a sterile cork borer (5 mm diameter) before adding enough inoculum of the 48 h old broth cultures of LAB to fill a bored hole and left to disperse. The plates were left to dry and set in an incubator at 35°C ±2°C for 24 h to observe clear zones. The antibacterial potential of the isolate was observed in the presence of a clear zone around the bored hole, without any growth. The degree of inhibition was measured using a meter ruler in millimeters.

2.3 Pathogenicity screening of isolates

Pathogenicity screening of isolates was performed by plating the LAB strains on sterile nutrient agar with 5% of human blood been added. The plates were incubated at 37°C for 24 h and the red blood cell lytic activity surrounding the LAB colonies was reported. The zones that were green around the colonies were regarded as alpha hemolysis, clear zones around the colonies were regarded as β-hemolysis, and those with no zones around were regarded as gamma hemolysis on the blood agar plates. Only strains with gamma hemolysis were considered safe for further use (Mangia et al., 2019).

2.4 Characterization of isolates

All isolates that passed pathogenicity screening were characterized using 16 S rRNA polymerase chain reaction (PCR) and Sanger partial gene sequencing.

Genomic DNA was extracted using a commercial bacterial DNA extraction kit following the manufacturer’s protocol. The 16S rRNA gene was amplified using universal primers 27F and 1492R. PCR amplification was carried out in a 25 μL reaction mixture containing template DNA (approximately 50 ng), PCR master mix, primers (0.5 μM each), and nuclease-free water Wang et al. (2011).

Amplification conditions were: Initial denaturation: 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min and final extension: 72°C for 7 min. PCR products were verified by agarose gel electrophoresis (1.5%) and visualized under UV illumination.

The PCR sequencing cocktail mixture comprised 10 μL 5x GoTaq colourless reaction, 3 μL of 25mM MgCl2, 1 μL of 10 mM mix of dNTPs, 1 μL of 27F 5′-AGA GTT TGA TCM TGG CTC AG-3′ and - 1525R, 5′-AAGGAGGTGATCCAGCC-3′ primers and 0.3 units of Taq DNA polymerase (Promega, USA). Up to 42 μL, the mixture was mixed with 8 μL of sterile distilled water DNA template.

PCR products were purified using ethanol precipitation and visualized on 1.5% agarose gel electrophoresis. Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI 3130xl Genetic Analyzer.

2.5 Preparation of cell-free supernatant

LAB cultures were grown in MRS broth at 37°C for 24 h and centrifuged at 4000 rpm for 10 min. The supernatant was collected and filtered using a 0.22 μm membrane filter to obtain cell-free supernatant (CFS). The pH of the CFS was adjusted to 6.5 using 1 M NaOH to eliminate the inhibitory effects of organic acids before antimicrobial testing. The cultures were centrifuged at 10,000 rpm for 10 min at 4°C to remove bacterial cells. The supernatant was collected and adjusted to pH 6.5 using 1 M NaOH to eliminate the inhibitory effect of organic acids. The neutralized supernatant was then filter sterilized using a 0.22 μm membrane filter and used as crude bacteriocin preparation (cell-free supernatant).

2.6 Optimization of cell-free supernatant and crude bacteriocins

2.6.1 Effect of incubation time

Pure cultures of LAB strains were transferred into MRS broth and incubated at 37°C for (24, 48, 72, 96, 120, 144, or 168) h, respectively. After incubation, centrifugation was done on the broth culture at 4,000 rpm for 10 min. The cell-free supernatant (CFS) was then transferred into sterile containers for antibacterial analysis against five selected bacterial pathogens (Bacillus subtilis, Bacillus proteolyticus, Pseudomonas aeruginosa, Klebsiella pneumonia, and Pseudomonas fuscoginae) using the agar well diffusion procedure and measurement of zones of inhibition, as described in Section 2.2.

2.6.2 Effect of pH

To determine the effect of pH, CFS/crude bacteriocin was adjusted to various pH levels of 3, 4, 5, 6, 7, 8, 9 and 10, respectively, using 1 M HCl and/or I M NaOH, and a pH meter was used to measure the pH level. An overnight broth culture of the bacterial pathogen was mixed with the prepared nutrient agar and poured into sterile petri dishes. A sterile cork borer was used to make wells of 6 mm diameter and loaded with 50 μL of the CFS or crude bacteriocin at various pH, then the plates were incubated for 24 h at 37°C, using the agar well diffusion procedure and measurement of zones of inhibition as described in Section 2.2.

2.6.3 Effect of organic solvents

The CFS/crude bacteriocins of each LAB strain were incubated with respective organic solvents (10%) which include acetone, ethanol, ethyl acetate and methanol. The antibacterial activity of CFS and crude bacteriocins was determined using the agar well diffusion procedure and measurement of zones of inhibition, as described in Section 2.2.

2.6.4 Effect of temperature

For The effect of temperature on CFS/crude bacteriocins was investigated by subjecting them to heat treatment at various temperatures at (30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 121°C) for 30 min, respectively. Following the treatment, all the CFS and crude bacteriocin samples went for a 24 h incubation period at 37°C. The antibacterial activity of CFS and crude bacteriocins was determined using the agar well diffusion procedure and measurement of zones of inhibition, as described in Section 2.2.

2.6.5 Effect of ultra-violet radiation

To determine the effect of UV radiation, cell-free supernatants/crude bacteriocins were exposed to ultraviolet irradiation for different durations (20, 40, 60, and 80 min, and no radiation). For UV treatment, samples were exposed in sterile Petri dishes with surface area 90 mm diameter.

The antibacterial activity of CFS and crude bacteriocins was determined using the agar well diffusion procedure and measurement of zones of inhibition, as described in Section 2.2.

2.6.6 Effect of storage conditions

The stability of the CFS/crude bacteriocins of LAB strains was observed under different storage conditions (freezing, refrigeration, and ambient conditions). The antibacterial potential of CFS and crude bacteriocins was determined using the agar well diffusion procedure and measurement of zones of inhibition, as described in Section 2.2.

2.6.7 Effect of proteases

The CFS/crude bacteriocins were treated with proteases (trypsin and pepsin at 1 mg/mL) to verify their protein structure as described by Zdolec et al. (2007). The proteases were prepared according to the manufacturer’s instructions. Following the treatment with pepsin and trypsin, the treated CFS was placed in an incubator for 30 min at 80°C. The antibacterial activity of CFS and crude bacteriocins was observed using the agar well diffusion procedure and measurement of zones of inhibition, as described in Section 2.2.

2.6.8 Effect of concentration

Crude bacteriocins were prepared at concentrations ranging from 10 to 100 mg/mL. Preparation of concentration: For 10 mg/mL, 420 μL of crude bacteriocin was mixed with 4.58 mL of sterile distilled water. The higher the concentration, the higher the mixture is utilized. The antibacterial activity of CFS and crude bacteriocins was determined using the agar well diffusion procedure and measurement of zones of inhibition, as described in Section 2.2.

2.7 Statistical analysis

All experiments were conducted in triplicate. Results are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS version 25. Differences among treatments were evaluated using one-way ANOVA followed by Tukey’s post-hoc test, with significance set at p < 0.05.

3.1 Test bacterial species

A total of 33 lactic acid bacteria (LAB) were isolated from Nigerian fermented foods (Iru, Ogi and fufu). Preliminary screening of the isolates for bacteriocin production yielded five LAB strains for a detailed study. Polymerase chain reaction and partial gene sequencing of the LAB strains revealed three strains of Lactobacillus plantarum, one strain of Lactobacillus paracasei and one strain of Lactobacillus acidophilus ( Table 1).

3.2 Effect on cell free supernatant (CFS)

Culture age

Generally, peak inhibitory effects occur between 48 h and 120 h, after which activity wanes. Early cultures (24 h) produced moderate inhibition, whereas very old cultures (144–168 h) generally showed sharp declines or complete loss of activity ( Table 2).

Among the test pathogens, K. pneumoniae and P. aeruginosa were the most frequently inhibited. In the case of B. subtilis, inhibition was intermittent and often zero beyond early time points, whereas P. fuscoginae and B. proteolyticus responses varied widely by LAB strain; for the LAB strains, L. plantarum (PV937063) showed the broadest spectrum, with strong inhibition (16–21 mm) against K. pneumoniae and P. aeruginosa at 24–72 h, with activity dropping markedly at 96 h but rebounding at 120 h for several of the pathogens ( Table 2).

In the case of L. paracasei (PV937064), a moderate early effect (24 h) with B. subtilis and B. proteolyticus was recorded with marked increases at 48–120 h, reaching up to 17 mm against P. aeruginosa and sustained activity against P. fuscoginae over 144 h. For the setup with L. plantarum (PV937062), a more selective activity was observed, showing the strongest and most consistent inhibition of P. fuscoginae and P. aeruginosa at 48 h and again at 120 h with little or no activity towards K. pneumoniae ( Table 2).

In addition, L. acidophilus (PV937061) showed the weakest overall activity with no detectable activity until 120 h, when modest zones (11 mm) appeared against K. pneumoniae, B. proteolyticus, and P. aeruginosa, then quickly declined. A narrow spectrum was observed with L. plantarum (PV937065), with early inhibition of K. pneumoniae and B. subtilis at 24 h (13 mm), and B. proteolyticus at 72 h ( Table 2).

pH

In general, the medium pH had a significant impact on the antimicrobial activity of the cell-free supernatant, with acidic to nearly neutral environments (pH 3–6) exhibiting higher inhibitory effects. Activity frequently decreased at alkaline levels (≥8), with some strains exhibiting abrupt decreases in inhibition against specific pathogens or total loss of inhibition ( Table 3).

Across the test pathogens, K. pneumoniae and P. aeruginosa were most consistently inhibited at the respective pH ranges, with zones of inhibition greater than 15 mm. When tested against P. fuscoginae and B. proteolyticus, the most varied and strain-dependent responses were observed at the respective pH values. However, substantial inhibition of B. subtiliswas observed at acidic and neutral pH.

Among the LAB strains, L. plantarum (PV937063) displayed the highest activity against the test pathogens, with zones of inhibition ranging from 13 to 22 mm. The observed inhibition in the presence of L. plantarum (PV937063) was evident across all pathogens and pH ranges, although the most stable activity was evident in acidic environments (pH 3–6). In addition, in terms of specific inhibition against a test pathogen, the highest activity was L. paracasei (PV937064) against K. pneumonia with maximal effects at pH 4-6, reaching 24 mm ( Table 3).

Organic solvents

Generally, all cell-free supernatants consistently showed no inhibitory activity against the test pathogens treated with ethyl acetate. The highest inhibitory activity was observed for ethanol-treated CFS, followed by methanol and, to a lesser extent, acetone ( Table 4).

Among the different CFS of the test LAB strains, K. pneumoniae was only inhibited by ethanol treatment. The highest levels of inhibition were observed in L. plantarum (PV937063, PV937062, and PV937065) (14.0 – 15 mm). Based on the activity of L. acidophilus (PV937061) and L. paracasei (PV937064) (12–14 mm), ethanol-treated CFS was observed to be the most potent inhibitor ( Table 4). Pseudomonas aeruginosa was inhibited by almost all LAB strains, particularly those treated with ethanol treatments of Lactobacillus plantarum (PV937063) and L. paracasei (PV937064), with inhibition zones of 19 mm ( Table 4).

Incubation temperature

In general, temperature affected the patterns of activity, with broad thermostability observed in some strains, whereas others displayed restricted or specific inhibition against the test pathogens. Among the test pathogens, P. fuscoginae, P. aeruginosa, and B. subtilis all showed similar inhibitory activities against L. plantarum (PV937063), with zones of inhibition that ranged from 10–15 mm at 30–60°C.

Generally, highest heat tolerance was observed at between 70 and 80°C, where inhibition against P. fuscoginae (14.5–15.0 mm) and P. aeruginosa (14.0 mm) was highest, although no inhibition against B. proteolyticus or K. pneumoniae. Among the CFS, the most selective trend was observed in L. paracasei (PV937064), with inhibition only against P. fuscoginae at the different temperature treatments, with highest zone of inhibition at 40°C (27.5 mm) and modest inhibition (10–13 mm) in other treatments ( Table 5).

On the other hand, L. plantarum (PV937062) showed strong inhibition against P. fuscoginae, P. aeruginosa, and B. proteolyticus at different temperature treatments, maintaining significant activity at sterilization (13.5–17.5 mm at 121°C). However, no activity was observed against B. subtilis. The highest inhibition was recorded for at 70°C and 80°C treatment with zones of inhibition 25.5 mm and 20 mm against K. pneumoniae and P. aeruginosa, respectively.

For L. acidophilus (PV937061), consistent and moderate inhibition was observed against most tested pathogens whileL. Plantarum (PV937065) showed lower range of antimicrobial activity at the respective temperature treatments, with strong activity against P. fuscoginae at 80°C (22.5 mm) and K. pneumoniae at 40°C (20 mm). However, activity was however less consistent at 121°C ( Table 5).

Ultra-violet (UV) radiation

Overall, the effect of ultra-violet (UV) irradiation on the antimicrobial activity of CFS showed distinct strain- and pathogen-specific responses. The highest inhibition was recorded in the presence of L. plantarum (PV937063), which showed strong activity against K. pneumoniae (20–30 mm), P. aeruginosa (20–30 mm), and B. subtilis (20–22 mm) after exposure to UV light for 60–80 min. However, no activity was observed against B. proteolyticus at after 40 min of UV exposure. The cell-free supernatant produced by Lacticaseibacillus paracasei exhibited reduced antibacterial activity following UV exposure ( Table 6).

In contrast, L. paracasei (PV937064) showed extreme sensitivity to UV treatment, with pathogen inhibition only noticeable during the first 40 min. In addition, L. plantarum (PV937062) showed remarkable inhibition against K. pneumoniae and P. aeruginosa (up to 21 mm), although no effect was observed against B. subtilis and B. proteolyticus. After 60 min of exposure, decreased activity was observed for L. plantarum (PV937065) against P. aeruginosa, whereas moderate inhibition was maintained against K. pneumoniae and B. proteolyticus (19–22 mm) ( Table 6).

Storage condition

Freezing and refrigerated storage conditions often resulted in partial or total loss of action, while ambient storage maintained the highest inhibition across the test pathogens ( Table 7).

Among the test pathogens, P. aeruginosa and P. fuscoginae were the most consistently inhibited, with inhibition zones sometimes exceeding 20 mm. This was followed by B. subtilis and K. pneumoniae, then B. proteolyticus which showed lesser or more varied inhibition ( Table 7).

Proteases

In general, treatment with pepsin completely eliminated the antibacterial activity against all strains and test pathogens. Although activity was also typically lost for most of the CFS, L. plantarum (PV937063) and L. paracasei (PV937064) retained little inhibition (10.5 mm) against P. aeruginosa ( Table 8).

3.3 Effect on crude bacteriocin

pH

Overall, acidic conditions (pH 3–4) did not affect the inhibitory activity of crude bacteriocins against the tested pathogens. However, as the pH increased towards neutral and alkaline conditions, inhibition generally decreased, and in several cases, the activity was terminated completely ( Table 9).

Among the tested strains, bacteriocins from L. plantarum (PV937063) showed the highest and most potent inhibitory activities, especially at pH 3 and 4, where the zones of inhibition reached 28–32 mm against K. pneumoniae and P. fuscoginae. The activity remained evident at near-neutral pH (pH 7). However, total loss of inhibition was observed against P. aeruginosa and B. subtilis, whereas at pH ≥ 9, activity against B. proteolyticus and B. subtilis was entirely decreased ( Table 9).

For the bacteriocin from L. paracasei (PV937064), medium but consistent inhibition against the test pathogens was observed, with the largest inhibition against B. proteolyticus at pH 3 (27.5 mm). For bacteriocin from L. plantarum (PV937062), more selective activity, particularly strong inhibition of P. fuscoginae (25 mm), was observed at pH 9. However, beyond pH 7, the activity against P. aeruginosa was absent ( Table 9).

In the presence of the bacteriocin from L. acidophilus (PV937061), the weakest overall activity was observed, with inhibitory effects restricted mainly to acidic pH, where zones reached 27.5 mm against B. proteolyticus and B. subtilis at pH 3. For L. plantarum (PV937065), medium and variable activity was observed against the test pathogens at the different pH treatments, with high activity at pH 8 against B. subtilis (25 mm) ( Table 9).

Organic solvents

In the presence of crude bacteriocin from L. plantarum (PV937063), ethyl acetate, methanol, and acetone treatments did not affect the antibacterial activity against K. pneumoniae, P. fuscoginae and P. aeruginosa. However, no inhibition was observed against B. pretolyticus when bacteriocin was treated with ethyl acetate, acetone, or methanol. Except for acetone treatment, all other solvent-treated bacteriocins from L. plantarum (PV937063) demonstrated inhibitory activity against B. subtilis ( Table 10).

In the case of the bacteriocin from L. plantarum (PV937063), the highest inhibition was observed against K. pneumoniae (24 mm) and P. aeruginosa (22.5 mm), while there was a complete loss of activity against B. proteolyticus. In contrast, ethanol treatment substantially inhibited B. proteolyticus (14.5 mm) and B. subtilis (16.5 mm). In addition, medium but consistent activity was observed for bacteriocin from L. paracasei (PV937064) in the different treatments. Although ethyl acetate yielded little or no inhibition against P. fuscoginae and P. aeruginosa, methanol and acetone increased its activity against P. fuscoginae and P. aeruginosa (17 mm), with activity observed against B. subtilis (16.5 mm) following ethanol treatment ( Table 10).

Incubation temperature

Generally, temperature significantly influenced the bacteriocin activity, with the greatest inhibition occurring between 30 and 40°C and a steady decrease at higher temperatures. Some bacteriocins from some of the strains showed thermostable features, retaining remarkable inhibition up to 121°C ( Table 11).

Among the LAB strains, bacteriocins from Lactobacillus plantarum (PV937063) consistently produced the most dominant and largest inhibition, especially against Klebsiella pneumoniae and Pseudomonas fuscoginae (16–19 mm at 30–40°C), with retained activity at 121°C (10–18 mm). Bacteriocin from L. paracasei (PV937064) also showed stable activity across most pathogens at the different temperature treatments, maintaining inhibition up to 121°C. In contrast, although bacteriocin from L. plantarum (PV937062) was highly effective against Bacillus subtilis (27.5 mm at 30°C), less stable inhibition was observed against other pathogens in the other treatments. Bacteriocin from L. acidophilus (PV937061) showed the lowest stability, with strong initial inhibition at 30°C against K. pneumoniae (17.5 mm) and B. proteolyticus (23 mm), but significantly reduced activity pathogens at elevated temperatures. For bacteriocin from L. plantarum (PV937065), activity was observed following treatment at 70°C (18–19 mm against K. pneumoniae and B. proteolyticus) and consistent at 121°C ( Table 11).

Ultra-violet radiation

In general, ultraviolet (UV) light exposure had a time-dependent effect on the test bacteriocins, with the extent of inhibition against pathogens occurring with longer exposure times. After 20 to 40 min of exposure, the inhibitory activity reached a maximum, followed by a significant decrease. Among the test pathogens, K. pneumoniae and P. aeruginosa were the most consistently inhibited across LAB strains, while activity against B. subtilis and B. proteolyticus varied more widely. The inhibition of P. fuscoginae was particularly unstable, showing a pronounced reduction with extended UV exposure ( Table 12).

Bacteriocin fromL. Plantarum (PV937063) showed the largest spectrum of activity, maintaining high inhibition against K. pneumoniae (19.5 mm at 20 min) and B. proteolyticus (22.5 mm at 20 min) before progressive decline in activity with exposure to UV. Notably, the inhibition of P. aeruginosa was the highest at 60 min (20 mm). In addition, L. paracasei (PV937064) demonstrated stable activity, with strong inhibition against P. fuscoginae (17.5 mm at 20 min) and B. subtilis (15–19 mm across treatments), though responses diminished significantly at 80 min ( Table 12).

In contrast, L. plantarum (PV937062) showed a limited but steady profile, showing moderate activity (10–16 mm) across most pathogens with less drastic fluctuations over time. For L. acidophilus (PV937061), the loss of inhibition of P. fuscoginae and P. aeruginosa beyond was 40 min. Although high inhibition of B. subtilis (22.5 mm at 20–60 min) and B. proteolyticus (16–18 mm) was observed for L. plantarum (PV937065) at shorter UV exposure times, the activity was reduced after 40 min of exposure ( Table 12).

Ultra-violet radiation

In general, ultraviolet (UV) light exposure had a time-dependent effect on the test bacteriocins, with the extent of inhibition against pathogens occurring with longer exposure times. After 20 to 40 min of exposure, the inhibitory activity reached a maximum, followed by a significant decrease. Among the test pathogens, K. pneumoniae and P. aeruginosa were the most consistently inhibited across LAB strains, while activity against B. subtilis and B. proteolyticus varied more widely. The inhibition of P. fuscoginae was particularly unstable, showing a pronounced reduction with extended UV exposure ( Table 12).

Bacteriocin from L. Plantarum (PV937063) showed the largest spectrum of activity, maintaining high inhibition against K. pneumoniae (19.5 mm at 20 min) and B. proteolyticus (22.5 mm at 20 min) before progressive decline in activity with exposure to UV. Notably, the inhibition of P. aeruginosa was the highest at 60 min (20 mm). In addition, L. paracasei (PV937064) demonstrated stable activity, with strong inhibition against P. fuscoginae (17.5 mm at 20 min) and B. subtilis (15–19 mm across treatments), though responses diminished significantly at 80 min ( Table 12).

In contrast, L. plantarum (PV937062) showed a limited but steady profile, showing moderate activity (10–16 mm) across most pathogens with less drastic fluctuations over time. For L. acidophilus (PV937061), the loss of inhibition of P. fuscoginae and P. aeruginosabeyond was 40 min. Although high inhibition of B. subtilis (22.5 mm at 20–60 min) and B. proteolyticus (16–18 mm) was observed for L. plantarum (PV937065) at shorter UV exposure times, the activity was reduced after 40 min of exposure ( Table 12).

Storage condition

Generally, the antibacterial activity of bacteriocins is significantly affected by storage conditions, with ambient and refrigerated conditions typically maintaining stronger inhibition than freezing conditions. Although refrigerated condition maintained potency and occasionally increased activity, activity was either decreased or lost with freezing condition ( Table 13).

For bacteriocin from L. plantarum (PV937063) highest spectrum, with strong inhibition against B. subtilis (27.0 mm refrigerated) and K. pneumoniae (26.5 mm refrigerated, 24.0 mm ambient) were recorded. In addition, significant activity against B. subtilis was maintained during ambient and refrigerated storage, while freezing resulted in a significant loss of activity. Under the respective storage conditions, L. paracasei (PV937064) consistently inhibited P. aeruginosa (16–22 mm) and P. fuscoginae (16–19 mm). However, a more selective activity was observed for bacteriocin from L. plantarum (PV937062), which inhibited P. fuscoginae (17–19 mm) and P. aeruginosa (13–21 mm), but showed little to no inhibition against K. pneumoniae. Under the respective storage conditions, L. acidophilus (PV937061) showed limited overall activity, losing all inhibition against K. pneumoniae and B. proteolyticus, while recording activity against P. aeruginosa (15–21 mm) and P. fuscoginae (19–20 mm). Although there was no activity against K. pneumoniae, bacteriocin from L. plantarum (PV937065) showed remarkable inhibition against P. aeruginosa (19–22 mm) and B. subtilis (21 mm ambient) ( Table 13).

Proteases

Following treatment with proteases, the inhibitory activity of the bacteriocins against the majority of pathogens was lost after pepsin treatment. The two exceptions were L. plantarum (PV937065), which demonstrated a narrow inhibitory impact against K. pneumoniae (10.0 mm), and L. plantarum (PV937063), which maintained moderate activity against B. proteolyticus (15.5 ± 6.36 mm) and B. subtilis (12.5 ± 3.54 mm). Following exposure to pepsin, L. paracasei (PV937064) also retained residual inhibition against B. subtilis (15.5 ± 6.36 mm) ( Table 14).

In contrast, more selective retention of activity across the strains was observed after trypsin treatment. For bacteriocin from L. acidophilus (PV937061) inhibition against K. pneumoniae (12.5 ± 3.54 mm), P. aeruginosa (16.0 ± 5.66 mm), and B. subtilis (12.5 ± 3.54 mm) was observed. In the case of L. plantarum (PV937063), inhibition against K. pneumoniae and P. aeruginosa was completely eliminated, but inhibition against P. fuscoginae (17.5 ± 3.54 mm) and B. subtilis (12.5 ± 3.54 mm) following trypsin treatment was observed. L. plantarum (PV937062) and (PV937065) also showed resistance to enzymatic degradation, retaining action only against some pathogens. This trend revealed that most of the protease-resistant bacteriocins were produced by L. plantarum (PV937063) and L. acidophilus (PV937061). In contrast, L. paracasei (PV937064) maintained a pathogen-limited action, whereas L. plantarum (PV937062) and (PV937065) showed a limited inhibitory range.

Bacteriocin concentration

The inhibition of K. pneumoniae steadily increased from 14.5 mm at 10 mg/mL to 24.0 mm at 100 mg/mL, with a similar gradual trend against P. aeruginosa (10.5–20.0 mm) for L. plantarum (PV937063). L. paracasei (PV937064) showed delayed but essential activity, particularly against P. fuscoginae, P. aeruginosa, and B. subtilis, with inhibition increasing sharply above 50 mg/mL and peaking at 19–24 mm at 100 mg/mL. In addition, L. plantarum (PV937062) showed a narrower spectrum, maintaining only steady inhibition (10–19 mm) against K. pneumoniae, P. fuscoginae, and B. subtilis ( Table 4). In addition, the widest concentration-dependent responses were observed for L. acidophilus (PV937061), which showed activity against all tested pathogens. In the case of L. plantarum (PV937065), a smaller but potent effect was observed, especially against K. pneumoniae (21 mm) and P. aeruginosa (18 mm) at 100 mg/mL ( Table 15).