Protease Sensitivity and Stress Adaptation of Bioactive Peptide-Producing Lactic Acid Bacteria: Functional Implications for Food Biopreservation

doi:10.21203/rs.3.rs-7542671/v1

Download PDF

Research Article

Protease Sensitivity and Stress Adaptation of Bioactive Peptide-Producing Lactic Acid Bacteria: Functional Implications for Food Biopreservation

https://doi.org/10.21203/rs.3.rs-7542671/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 25 Nov, 2025

Read the published version in BMC Biotechnology →

You are reading this latest preprint version

Background

The search for safe, natural food preservatives has turned attention to antimicrobial peptide (AMP)-producing lactic acid bacteria (LAB). These AMPs, which are bioactive peptides of proteinaceous nature, inhibit a broad spectrum of foodborne pathogens. Their proteinaceous composition ensures safety and digestibility; however, their effectiveness depends on the physiological resilience of the producing LAB under food-relevant stresses and the susceptibility of the AMPs to proteolytic degradation.

Results

The 13 AMP-producing LAB strains tolerated a wide pH range (4.5–8.5), multiple temperatures (20–45°C), and moderate to high salt concentrations (5.5–10% NaCl), demonstrating robustness under diverse food processing and storage conditions. Even after exposure to these physiological stresses, the strains retained antimicrobial activity, producing zones of inhibition ranging from 5 mm under extreme stress conditions to 20 mm under optimum growth conditions against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. The antimicrobial peptides were completely inactivated by protease treatments with pepsin, trypsin, proteinase K, and papain, confirming their proteinaceous nature while highlighting protease susceptibility. Six of the 13 LAB strains had been previously 16S rRNA-sequenced (GenBank accession numbers PV983358–PV983363), including one novel strain showing 93.54% sequence identity to the closest known species; this strain is currently undergoing whole-genome sequencing, underscoring the rigor of prior molecular characterization and the potential for discovering novel antimicrobial producers.

Conclusions

AMP-producing LAB from Nigerian non-dairy fermentations exhibit broad physiological adaptability and produce proteinaceous antimicrobial peptides with notable inhibitory activity against foodborne pathogens, even under stress conditions. Although complete protease susceptibility limits in vivo stability, their safety, traceable identification, and environmental robustness underscore their promise as natural, clean-label food preservatives, supporting the development of safe, minimally processed food strategies.

Protease sensitivity

Stress adaptation

Bioactive peptide-producing bacteria

Lactic acid bacteria (LAB)

Food biopreservation

Antimicrobial activity

Functional properties

The growing consumer demand for safe, natural, and minimally processed foods has spurred considerable interest in microbial-derived bio-preservatives, which offer an alternative to synthetic chemical additives that may pose health concerns or alter sensory properties [1, 2, 3]. Among these natural preservatives, antimicrobial peptide (AMP)-producing lactic acid bacteria (LAB) have emerged as particularly promising due to their dual functionality: they inhibit a broad spectrum of foodborne pathogens and spoilage organisms, while remaining digestible and generally recognized as safe (GRAS) [4, 5, 6, 7].

The proteinaceous nature of AMPs confers both safety and specificity, as they are typically degraded by digestive enzymes and do not accumulate in the body. In addition, AMPs exhibit diverse mechanisms of action, including disruption of cell membranes, interference with intracellular targets, and inhibition of essential enzymatic processes, which collectively enable them to target both Gram-positive and Gram-negative bacteria [8, 9, 10, 11]. This broad-spectrum antimicrobial activity, coupled with their environmental resilience, makes AMP-producing LAB ideal candidates for incorporation into minimally processed foods, where they can enhance safety and extend shelf life without compromising nutritional quality or organoleptic properties [12, 13, 14].

However, the effectiveness of AMPs in food systems is strongly influenced by the physiological resilience of the producing LAB, as environmental conditions such as pH fluctuations, temperature variations, and high salt concentrations can impact both microbial growth and AMP production [8, 15, 16, 17, 18]. Furthermore, AMPs are often susceptible to proteolytic degradation, which may limit their functionality in protein-rich or enzyme-active foods [19, 20, 21, 22, 23].

While several studies have focused on AMP-producing LAB from dairy products, non-dairy fermented foods remain an underexplored source, particularly in Africa [24, 25]. These substrates, often produced through spontaneous fermentation, harbor diverse microbial consortia that adapt to varying environmental and nutritional conditions, thereby providing a unique niche for the evolution of physiologically resilient strains [26, 27, 28].

Nigerian non-dairy fermented foods such as soy milk, tigernut milk, and sorghum-based gruels are not only staple components of local diets but are also characterized by rich microbial diversity shaped by traditional processing methods [29, 30]. This makes them a promising reservoir for LAB strains capable of producing antimicrobial peptides with broad activity and stability under food-relevant conditions [13, 31, 32]. Exploring these traditional fermentations could therefore expand the repertoire of biopreservative candidates, especially those that combine safety, adaptability, and inhibitory activity against foodborne pathogens [33, 34, 35, 36, 37].

This study evaluates the physiological adaptability and protease sensitivity of 13 AMP-producing LAB strains isolated from Nigerian non-dairy fermentations. By investigating their tolerance to food-relevant stresses and the stability of their AMPs, this work aims to assess their potential as natural, clean-label food preservatives, building on our prior molecular characterization that identified six strains, including a putative novel species.

The workflow of this study illustrates the stepwise approach used to investigate thirteen AMP-producing lactic acid bacteria (LAB) from Nigerian non-dairy fermentations (Fig. 1). The LAB strains were cultured and assessed for physiological adaptation under varying temperatures (20°C, 37°C, 45°C), pH values (4.5–8.5), and NaCl concentrations (5.5–10%). Their antimicrobial activity was evaluated using cell-free supernatants via agar well diffusion assays, followed by protease sensitivity testing. The workflow concludes with data analysis to determine the potential of these LAB strains for food biopreservation.

Bacterial strains and culture conditions

Thirteen AMP-producing LAB strains (SGA2, SGA12, SGB13, TNA7, TNA9, TNA10, TNA14, SGB11, SMA1, SMA12, SGA8, SMB5, and TNA4) were used in this study. These strains were previously isolated from Nigerian non-dairy fermented foods, including soy milk, tigernut milk, and sorghum gruel. Six of the strains were molecularly characterized in our earlier study through 16S rRNA sequencing (GenBank accession numbers PV983358–PV983363), including one putative novel strain currently undergoing whole-genome sequencing. All strains were maintained on de Man, Rogosa, and Sharpe (MRS) agar and sub-cultured in MRS broth at 37°C for 24 h prior to experimentation [38].

Physiological stress assays

The physiological adaptability of the LAB strains was evaluated under food-relevant stresses, including variations in pH, temperature, and salt concentration, with methods adapted from [39] with slight modifications. Briefly, actively growing cultures (5% v/v inoculum, 250 µL into 5 mL MRS broth) were incubated under each stress condition for 24 h.

For pH tolerance, MRS broth was adjusted to pH 4.5, 5.5, 6.5, and 8.5 using HCl or NaOH before inoculation, and cultures were incubated at 37°C. For temperature tolerance, inoculated MRS broth was incubated at 20°C, 37°C and 45°C. For salt tolerance, MRS broth was supplemented with NaCl at final concentrations of 5%, 6.5% and 10% (w/v) and incubated at 37°C.

Bacterial growth under all conditions was monitored spectrophotometrically (L7 Double Beam UV-VIS) at OD₆₀₀ after 0, 3, and 6 h of incubation.

Antimicrobial activity assays

Preparation of cell-free supernatants from 13 AMP-producing LAB strains

To obtain cell-free supernatants (CFS), actively growing antimicrobial peptide-producing LAB cultures were prepared by inoculating 1% (v/v; 0.1 mL/10 mL) of overnight culture into fresh MRS broth (ReadyMED, India) and incubating anaerobically at 37°C for 18–24 h. Cultures were centrifuged at 6000 rpm for 15 min (Eppendorf Centrifuge 5702) to remove cells, and the resulting supernatants were passed through 0.22 µm syringe filters (mdi Membrane Technologies, USA). The pH was adjusted to 6.5–7.0 using 1 M NaOH, and the filtrates were stored at − 20°C until further use [38].

Agar well diffusion assay

The inhibitory activity of LAB strains against foodborne pathogens was assessed after exposure to physiological stress conditions using Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 as test organisms. Pathogens were grown in Mueller–Hinton broth at 37°C for 18 h, and the inocula were standardized to 0.5 McFarland (≈ 1.5 × 10⁸ CFU/mL). Standardized suspensions were spread on Nutrient agar plates, after which wells (6 mm diameter) were created with a sterile cork borer and filled with 100 µL of cell-free supernatant (CFS) from the LAB strains. Plates were held at room temperature for 2 h to allow diffusion and then incubated at 37°C for 18–24 h. Antimicrobial activity was determined by measuring the diameter of clear zones surrounding the wells, with inhibition zones ≥ 0.5 mm considered positive [40]. To evaluate the effect of stress on antimicrobial activity, inhibition zones obtained under extreme stress conditions (corresponding to minimal growth) were compared with those recorded under optimum conditions.

Protease sensitivity tests

The proteinaceous character of the antimicrobial metabolites was examined by subjecting the cell-free supernatants (CFS) obtained from the 13 LAB strains to enzymatic degradation. The procedure was adapted from [41] with minor modifications. Specific enzyme–buffer systems were prepared as follows: trypsin (Sigma, Germany) in 40 mM Tris–HCl buffer (pH 8.2), pepsin (Merck, Germany) in 0.002 M HCl (pH 2.0), proteinase K (Sigma, Germany) in 10 mM Tris–HCl (pH 7.5), and papain (Sigma, Germany) in phosphate-buffered saline (PBS, pH 6.8). Equal volumes (1 mL) of CFS and the respective enzyme solution were combined, while buffer-only mixtures with CFS served as enzyme-free controls. Untreated AMPs were included as positive controls. After incubation at 37°C for 2 h, the enzyme activity was terminated by heating the mixtures in a boiling water bath for 5 min. The treated supernatants were subsequently tested for antimicrobial activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 using the agar well diffusion method as outlined earlier. The disappearance of inhibitory zones following enzyme exposure was taken as confirmation that the active compounds were proteinaceous in nature.

Statistical analysis

All assays were conducted in triplicate, and results are expressed as mean ± standard deviation. Variations in inhibition zones among strains and treatment conditions were assessed using one-way analysis of variance (ANOVA) with Tukey’s HSD post hoc test at a significance level of p < 0.05. Statistical analyses were performed in R version 4.5.1 using the agricolae package, while graphical representation of data, including bar plots with error bars and significance annotations, was generated with ggplot2 and ggpubr packages [42].

Antimicrobial peptide-producing bacterial strains

Thirteen AMP-producing LAB strains (SGA2, SGA12, SGB13, TNA7, TNA9, TNA10, TNA14, SGB11, SMA1, SMA12, SGA8, SMB5, and TNA4) were previously shown to exhibit antimicrobial activity against four test pathogens in our earlier study. In the present work, these strains were subjected to physiological stress conditions (temperature, pH, and NaCl) as well as protease sensitivity assays to evaluate the stability and proteinaceous nature of their antimicrobial peptides. Their antimicrobial activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 was further confirmed following exposure to stress conditions.

Effect of physiological stress on AMP-producing LAB

The physiological adaptability of the thirteen AMP-producing LAB strains was evaluated under different physiological stress conditions, including variations in NaCl concentration, pH, and temperature.

Growth of AMP-producing LAB strains at different temperatures

The growth of the thirteen AMP-producing LAB strains was evaluated at different incubation temperatures (20°C, 37°C, and 45°C) to assess their tolerance and activity under thermal stress (Fig. 2a-c).

At 20°C, the AMP-producing LAB strains exhibited relatively low initial OD values at 0 h, ranging from 0.03 (TNA7) to 0.203 (TNA4), indicating minimal growth at inoculation. By 3 h, all isolates demonstrated increased OD values (0.272–0.456), suggesting active growth. After 6 h, further increases were observed, with SGA8 (0.693) and SMB5 (0.695) showing the highest growth, whereas TNA7 (0.522) remained the least responsive.

At 37°C, initial OD values at 0 h ranged from 0.0067 (SGB13) to 0.5893 (SMA12). Growth was evident by 3 h across most isolates, with OD values between 0.4953 (SGA2) and 0.7467 (SMB5). SMA12 and TNA4 showed pronounced increases, while SGB13 and SGA12 displayed moderate growth. After 6 h, SMA12 reached the highest OD (0.896), followed by TNA4 (0.875) and SMB5 (0.794). In contrast, SGA12 and TNA10 showed a decline, with OD values dropping to 0.335 and 0.324, respectively.

At 45°C, OD values at 0 h were generally low, ranging from 0.0137 (TNA9) to 0.5427 (SMA1). By 3 h, all isolates had increased OD, with values between 0.184 (TNA9) and 0.6403 (SMA1). After 6 h, most isolates continued to grow, with TNA10 showing the highest OD (0.7063), followed by SGA2, SGA12, and TNA7 (≈0.620). SMA12 (0.589) and TNA4 (0.643) also exhibited considerable growth, while TNA9 showed the lowest response, reaching 0.4103 despite its increase compared to baseline.

Growth of AMP-producing LAB under different NaCl concentrations

The thirteen AMP-producing LAB strains exhibited variable growth at different salt concentrations (Fig. 3a-c). At 5% NaCl, initial OD values at 0 h ranged from 0.069 (TNA10) to 0.688 (TNA14), with other isolates such as SGA2 and SGB13 showing values around 0.145 and 0.132, respectively. After 3 h, all isolates demonstrated increased growth, with OD values between 0.4087 (TNA10) and 0.9757 (TNA14). By 6 h, further growth was observed, with TNA4 showing the highest OD (1.0193), followed by SMA12 (0.9667) and TNA14 (0.9413). Other isolates, including SGA2, TNA7, and SGA8, had OD values ranging from 0.7673 to 0.8583, while TNA10 remained the least responsive (0.6303).

At 6.5% NaCl, initial OD values were low, ranging from 0.033 (SGA2) to 0.1497 (SMA12 and SMB5), with other strains such as SGB13, TNA7, and TNA14 between 0.0597 and 0.0827. Growth after 3 h increased across most isolates, with TNA9 showing the largest rise (0.8587) and SGB13 the smallest (0.1257). At 6 h, OD values continued to increase moderately for most strains, with TNA7 (0.3411), SGB13 (0.3295), and TNA14 (0.3528) reaching the highest values. Some isolates, including SGA2, SGA12, and SMA12, showed smaller increases (0.2429–0.3221), while TNA9 and TNA10 slightly declined to 0.2415 and 0.2406, respectively.

At 10% NaCl, initial growth at 0 h was generally low, with OD values ranging from 0.0667 (TNA14) to 0.1857 (SGA12), and other strains such as SGB13, TNA7, and SMA1 between 0.0737 and 0.1497. By 3 h, most isolates exhibited increased growth, with values between 0.1213 (SMB5) and 0.6293 (SGB11). At 6 h, further growth was recorded, with TNA14 showing the highest OD (0.6493) followed by SGB11 (0.6293). Strains like SGA12, TNA7, and TNA9 displayed moderate increases (0.3693–0.5293), whereas TNA10 and SGA8 showed the smallest growth increments (0.2583–0.2763).

Growth of AMP-producing LAB strains at different pH

The growth of the thirteen AMP-producing LAB strains varied across different pH conditions (Fig. 4a-d). At pH 4.5, initial optical density (OD) values at 0 h ranged from 0.017 (SGA8) to 0.2 (SGB11), indicating minimal starting growth. After 3 h, OD values increased in most isolates, from 0.135 (TNA10) to 0.452 (TNA4), reflecting active growth. By 6 h, OD values continued to rise, with SGA12 reaching 0.635 and SGB13 at 0.209, demonstrating further growth or metabolic activity.

At pH 5.5, initial OD values ranged from 0.051 (TNA10) to 0.670 (TNA14), with other isolates such as SGA2, SGB11, and SMA12 showing moderate initial growth (0.114–0.52). After 3 h, all strains exhibited increased OD values (0.291–0.828), indicating active growth, with SGA2 and TNA14 showing particularly strong growth. At 6 h, TNA14 recorded the highest OD of 0.981, followed by SGA2 at 0.943. Other isolates, including SGB13, TNA9, and SMB5, showed moderate increases (0.669–0.851), while TNA10 exhibited the lowest growth (0.606).

Under pH 6.5, initial OD values ranged from 0.1007 (TNA14) to 0.2317 (SGA2), with SGA12, SGB13, and TNA7 between 0.1047 and 0.1667. By 3 h, OD values had risen in most strains (0.276–0.609), and by 6 h, SGA2 reached the highest OD of 0.6337, followed by TNA14 (0.8287). Moderate growth was observed in TNA7, SGB11, and SMA1 (0.5607–0.7457), while SMA12 showed the lowest OD (0.5607).

At pH 8.5, starting OD values were low (0.0243–0.1333), with TNA7 at the minimum and TNA4 at the maximum. After 3 h, most strains exhibited increased growth (0.0453–0.6033), and by 6 h, TNA14 reached the highest OD of 0.6413, followed by SGA12 at 0.3613. Other isolates, including SGB13, TNA7, and TNA4, showed moderate growth (0.2353–0.4933). Overall, growth trends were generally consistent, although some strains showed slight declines or stabilization by the 6 h time point.

Antimicrobial activity of 13 AMP-producing LAB strains under physiological stress on S. aureus and E. coli

To evaluate the resilience of antimicrobial peptides (AMPs) produced by lactic acid bacteria (LAB) under physiological stress, we assessed their activity against Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. The LAB strains were subjected to stress conditions including varying temperature, pH, and NaCl concentrations. Heat map analysis revealed strain-specific differences, highlighting the variable capacity of individual LAB strains to maintain inhibitory activity under these stress conditions (Figs. 5 & 6).

Heat map analysis of antimicrobial activity of 13 LAB strains under temperature, pH and NaCl stress on S. aureus and E. coli

Heat map analysis revealed strain-specific variations in the antimicrobial activity of LAB-derived AMPs under thermal stress. At 37°C, most LAB-derived AMPs maintained strong inhibitory activity, with inhibition zones ranging from 13.00–19.33 mm against S. aureus and 13.00–17.33 mm against E. coli. Exposure to elevated temperature (45°C) led to a notable decline, with inhibition zones reduced to 7.33–11.33 mm for S. aureus and 5.33–9.33 mm for E. coli. At lower temperature (20°C), activity was moderately reduced, ranging from 9.33–13.67 mm for S. aureus and 7.33–11.67 mm for E. coli. Strain-specific differences were observed, with certain LAB strains (SGA12, SGA2, TNA9, TNA14, SMA12, SGB13) producing AMPs that retained higher activity under thermal stress, whereas others (TNA4, SGA8) were comparatively more sensitive.

The effect of pH on the antimicrobial activity of 13 physiologically stressed LAB strains was also evaluated. The peptides retained their inhibitory function across the tested pH range (4.5–8.5), demonstrating stability under both acidic and slightly alkaline conditions. At pH 6.5, most LAB-derived AMPs exhibited strong activity, with inhibition zones of 15.33–19.67 mm against S. aureus and 13.33–17.67 mm against E. coli. Exposure to higher pH (8.5) led to a marked decline in activity, ranging from 5.33–9.33 mm for S. aureus and 3.33–7.67 mm for E. coli, whereas at lower pH (4.5–5.5), inhibition zones ranged from 9.67–16.67 mm for S. aureus and 7.67–14.67 mm for E. coli. Positive controls (untreated AMPs) maintained full activity, confirming that the antimicrobial function of the peptides was inherently stable across these pH levels.

The effect of NaCl on the antimicrobial activity of 13 physiologically stressed LAB strains was also assessed. The peptides retained their inhibitory function across the tested NaCl concentrations (5.5–10%), demonstrating stability under both low and high osmotic stress. At 5.5% NaCl, most LAB-derived AMPs exhibited strong activity, with inhibition zones ranging from 11.33–15.67 mm against S. aureus and 9.33–13.67 mm against E. coli. At 6.5% NaCl, activity was moderately reduced, with inhibition zones of 9.67–13.67 mm for S. aureus and 7.67–11.67 mm for E. coli. Exposure to 10% NaCl resulted in a further decline, with inhibition zones of 6.67–10.67 mm for S. aureus and 4.67–8.67 mm for E. coli.

Protease sensitivity of AMPs

The antimicrobial activity of all the thirteen LAB-derived AMPs was completely abolished following treatment with proteinase K, trypsin, pepsin and papain. This indicates that the active compounds are entirely proteinaceous in nature. Untreated controls retained full activity, confirming that the observed inhibition was due to the AMPs (Table 1).

Table 1. Effect of protease treatment on the antimicrobial activity of 13 LAB-derived AMPs against S. aureus ATCC 25923 and E. coli ATCC 25922

“–” = no inhibition (activity abolished after protease treatment)

“+” = inhibition retained (untreated control showing full activity)

All 13 LAB-derived AMPs were tested against S. aureus ATCC 25923 and E. coli ATCC 25922, and results were consistent for both organisms.

This study evaluated the stability and functionality of antimicrobial peptides (AMPs) produced by thirteen LAB strains under different physiological stress conditions and protease treatments. The observed antimicrobial activity against Staphylococcus aureus and Escherichia coli even after exposure to temperature, pH, and NaCl stress highlights the resilience of these peptides and their potential application as natural biopreservatives in food systems, consistent with previous reports demonstrating the robustness of LAB-derived AMPs under environmental fluctuations [43, 44, 45, 46, 47]. The sensitivity of the antimicrobial activity to proteolytic enzymes further confirmed the proteinaceous nature of the bioactive compounds, in agreement with studies showing that bacteriocins and related LAB peptides are inherently proteinaceous [48, 49, 50, 51].

The ability of AMPs to retain inhibitory activity under moderate stress conditions is noteworthy, given that food processing and storage often expose bioactive compounds to fluctuating thermal, pH, and osmotic environments. Our findings are in line with earlier studies reporting that bacteriocins and other LAB-derived peptides generally exhibit remarkable stability to heat and pH variations [52, 53, 54, 55, 56, 57]. However, variability among strains suggests that specific peptide structures may contribute differently to stress tolerance, indicating potential for strain-specific applications in food preservation and safety enhancement.

Temperature strongly influences the growth and metabolite production of LAB. In this study, most strains grew optimally at 37°C, with SMA12, TNA4, and SMB5 showing consistently higher OD values after 6 h, consistent with reports that mesophilic LAB display maximal activity near body temperature, conditions relevant for food fermentation and health applications [58, 59, 60, 61].

Notably, strains such as TNA10, SGA2, and TNA7 maintained considerable growth at 45°C, reflecting thermotolerance comparable to Lactobacillus plantarum and Enterococcus faecium, which retain bacteriocin activity at elevated temperatures [62, 63, 64, 65]. Such resilience is advantageous for industrial environments where temperature fluctuations occur. In contrast, reduced growth at 20°C, particularly in TNA7, suggests limited psychrotolerance, although strains like SGA8 and SMB5 exhibited relatively higher activity, indicating potential for chilled food preservation [66. 67, 68, 69].

The strain-specific variability observed likely reflects differences in stress-response systems, including heat-shock proteins, membrane adaptation, and regulation of bacteriocin gene clusters [70, 71, 72]. Collectively, these findings highlight isolates with dual mesophilic and thermophilic adaptability as promising candidates for targeted food biopreservation.

The growth of AMP-producing LAB strains under different NaCl concentrations revealed distinct patterns, indicating varying degrees of salt tolerance among the isolates. At 5% NaCl, all strains demonstrated significant growth, with OD values increasing from 0.069–0.688 at 0 hours to 0.630–1.019 at 6 hours. Notably, TNA4, SMA12, and TNA14 exhibited the highest growth rates, suggesting a robust adaptation to moderate salinity levels. Conversely, TNA10 showed the least growth, indicating a lower tolerance to salt stress.

At 6.5% NaCl, initial OD values were lower, ranging from 0.033 to 0.1497. After 6 hours, growth increased moderately, with TNA7, SGB13, and TNA14 reaching OD values of approximately 0.33–0.35. Some strains, including SGA2, SGA12, and SMA12, exhibited minimal growth, highlighting their limited adaptability to higher salinity conditions.

At 10% NaCl, initial growth was minimal, with OD values between 0.0667 and 0.1857. Over 6 hours, growth remained subdued, with TNA14 and SGB11 achieving the highest OD values of 0.629 and 0.649, respectively. TNA10 and SGA8 showed negligible growth, underscoring their sensitivity to high salt concentrations.

These findings are consistent with previous reports on LAB salt tolerance. For example, [73] noted that bacterial isolates from saline environments are promising candidates for the fermentation of saline substrates, allowing high product yields without the need for prior desalination. Similarly, [74] demonstrated that certain LAB strains, such as Lactobacillus acidophilus CM1 and Lactobacillus delbrueckii OS1, can survive at 4–6% NaCl by synthesizing compatible solutes and modifying their membrane properties, reflecting their ability to adapt to high salinity.

The observed variability in growth responses among the LAB strains suggests that salt tolerance is a strain-specific trait, influenced by genetic and physiological factors. This variability has implications for the selection of LAB strains in food fermentation processes, particularly in products subjected to high salinity conditions [76, 77, 78].

The growth patterns of AMP-producing LAB under varying NaCl concentrations underscore the importance of strain selection based on salt tolerance for optimizing fermentation processes. Further studies are warranted to elucidate the molecular mechanisms underlying salt tolerance in LAB and to explore the potential of these strains in producing antimicrobial peptides under saline conditions.

The thirteen AMP-producing LAB strains exhibited variable growth across different pH conditions (4.5–8.5), reflecting strain-specific pH tolerance (Fig. 4a–d). Generally, growth was minimal at pH 4.5 and 8.5, while optimal growth occurred at pH 5.5–6.5, with TNA14 and SGA2 showing the highest ODs. These trends are consistent with previous studies indicating that LAB maintain metabolic activity within a narrow pH range, with acid or alkaline stress reducing growth and activity [72, 75, 79, 80]. The variability among strains suggests that pH tolerance is strain-specific, likely influenced by membrane adaptation and acid-resistance mechanisms. Understanding these differences is important for selecting robust LAB strains for fermentation processes and antimicrobial peptide production under variable pH conditions.

Heat map analysis revealed strain-specific differences in the antimicrobial activity of LAB-derived AMPs under temperature, pH, and NaCl stress on S. aureus and E. coli respectively (Figs. 5 & 6). Most AMPs maintained strong activity at 37°C, but activity declined at 45°C and moderately at 20°C, with strains SGA12, SGA2, TNA9, TNA14, SMA12, and SGB13 showing higher resilience.

Across pH 4.5–8.5, maximal activity occurred at pH 6.5, moderate activity at acidic pH, and reduced activity at alkaline pH, consistent with previous reports that LAB-derived AMPs function optimally under slightly acidic to neutral conditions [24, 81, 82]. AMPs also remained active across NaCl concentrations of 5.5–10%, though inhibition declined progressively with increasing salinity, in line with findings that osmotic stress can reduce AMP efficacy [83, 84].

These results demonstrate that several LAB strains produce AMPs with notable stability under thermal, pH, and osmotic stresses, highlighting their potential for food preservation and industrial applications [85, 86].

The complete loss of antimicrobial activity of all thirteen LAB-derived AMPs upon treatment with proteinase K, trypsin, pepsin, and papain confirms that the active compounds are entirely proteinaceous. Untreated controls retained full activity, verifying that inhibition was specifically due to the peptides. These findings are consistent with previous reports showing that bacteriocins and other LAB-derived antimicrobial peptides are protein-based and are inactivated by proteolytic enzymes [87, 88, 89] Such protease sensitivity is a key characteristic for confirming the proteinaceous nature of AMPs and differentiating them from non-protein antimicrobials like organic acids or hydrogen peroxide.

The complete inactivation of all AMPs by protease treatment not only confirms their proteinaceous nature and safety for food use, but also highlights a critical biotechnology opportunity to enhance their applicability through protective delivery strategies such as encapsulation, nano-formulation, or immobilization [90].

This study demonstrates that antimicrobial peptide (AMP)-producing lactic acid bacteria (LAB) isolated from Nigerian non-dairy fermentations exhibit distinct physiological adaptations to temperature, pH, and osmotic stress, with strain-specific variations in growth and peptide activity. Several strains maintained robust antimicrobial activity under thermal, acidic, alkaline, and high-salinity conditions, highlighting their potential for use in food biopreservation. The complete loss of activity upon protease treatment confirms the proteinaceous nature of the AMPs, reinforcing their classification as bacteriocins. Collectively, these findings underscore the promise of these LAB strains as stable, natural antimicrobial agents for enhancing food safety and shelf-life in diverse processing environments.

Author contributions

P.O.A. conceptualised the study and designed the experiments, A.O.O., O.A.A. supervised the research. P.O.A. performed the experiments, analysed the data, and drafted the manuscript. P.O.A. discussed the results. P.O.A., A.O.O., O.A.A. reviewed and edited the manuscript. All authors read and approved the final manuscript.

Funding

This research received no external funding.

Data availability

The 16S rRNA gene sequences of the six AMP-producing LAB isolates used in this study are deposited in the NCBI GenBank repository under accession numbers PV983358–PV983363. Additional datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Genetics, Genomics and Bioinformatics Department, National Biotechnology Research and Development Agency (NBRDA), Abuja, Nigeria

²Department of Microbiology, University of Abuja, Abuja, Nigeria

³College of Basic Sciences, Lagos State University of Science and Technology, Ikorodu, Lagos, Nigeria

⁴Department of Microbiology, Kaduna State University, Kaduna, Nigeria

Athar, H., Rafaqat, S., Habiba, U. E., Khan, Z. Z., Tirmazi, S. M. B., Noor, A. & Abdullah, M. (2025). Exploration of Natural Preservative Systems for Enhancing Microbial Stability and Shelf Life in Dairy-Based Food Matrices. Sch Acad J Biosci, 5, 602-613. https://doi.org/10.36347/sajb.2025.v13i05.014.
Nie, X., Zuo, Z., Zhang, R., Luo, S., Chi, Y., Yuan, X., ... & Wu, Y. (2025). New advances in biological preservation technology for aquatic products. npj Science of Food, 9(1), 15. https://doi.org/10.1038/s41538-025-00372-4
Karim, S. N., Hew, P. S., Anwar, F., Sukor, R., Jambari, N. N., Sanny, M., & Khatib, A. (2025). Innovative Technological Approaches in the Detection and Mitigation of Food Toxicants. Food Reviews International, 1-45. https://doi.org/10.1080/87559129.2025.2544980
Bisht, V., Das, B., Hussain, A., Kumar, V., & Navani, N. K. (2024). Understanding of probiotic origin antimicrobial peptides: a sustainable approach ensuring food safety. npj Science of Food, 8(1), 67. https://doi.org/10.1038/s41538-024-00304-8.
Akpoghelie, P. O., Edo, G. I., Mafe, A. N., Isoje, E. F., Igbuku, U. A., Ali, A. & Alamiery, A. A. (2025). Food, health, and environmental impact of lactic acid bacteria: The superbacteria for posterity. Probiotics and Antimicrobial Proteins, 1-37. https://doi.org/10.1007/s12602-025-10546-x.
Nebbia, S., Lamberti, C., Lo Bianco, G., Cirrincione, S., Laroute, V., Cocaign-Bousquet, M., & Pessione, E. (2020). Antimicrobial potential of food lactic acid bacteria: bioactive peptide decrypting from caseins and bacteriocin production. Microorganisms, 9(1), 65. https://doi.org/10.3390/microorganisms9010065.
Corrêa, J. A. F., de Melo Nazareth, T., Rocha, G. F. D., & Luciano, F. B. (2023). Bioactive antimicrobial peptides from food proteins: Perspectives and challenges for controlling foodborne pathogens. Pathogens, 12(3), 477. https://doi.org/10.3390/pathogens12030477.
Saubenova, M., Rapoport, A., Yermekbay, Z., & Oleinikova, Y. (2025). Antimicrobial Peptides, Their Production, and Potential in the Fight Against Antibiotic-Resistant Pathogens. Fermentation, 11(1), 36. https://doi.org/10.3390/fermentation11010036.
Volzing, K., Borrero, J., Sadowsky, M. J., & Kaznessis, Y. N. (2013). Antimicrobial peptides targeting Gram-negative pathogens, produced and delivered by lactic acid bacteria. ACS synthetic biology, 2(11), 643-650. https://doi.org/10.1021/sb4000367.
Abedin, M. M., Chourasia, R., Phukon, L. C., Sarkar, P., Ray, R. C., Singh, S. P., & Rai, A. K. (2024). Lactic acid bacteria in the functional food industry: Biotechnological properties and potential applications. Critical Reviews in Food Science and Nutrition, 64(29), 10730-10748. https://doi.org/10.1080/10408398.2023.2227896.
Liu, S., Brul, S., & Zaat, S. A. (2020). Bacterial persister-cells and spores in the food chain: Their potential inactivation by antimicrobial peptides (AMPs). International Journal of Molecular Sciences, 21(23), 8967. https://doi.org/10.3390/ijms21238967.
Mihooliya, K. N., & Kumari, A. (2024). Bioprocessing and market aspects of antimicrobial peptides. In Evolution of antimicrobial peptides: From self-defense to therapeutic applications (pp. 167-197). Cham: Springer Nature Switzerland. https://doi.org/10.1007/978-3-031-67515-7_7.
Kumar, P., Singh, S., Sankhyan, S., & Ray, S. (2024). Metabolic Engineering of Lactic Acid Bacteria for Antimicrobial Peptides Production. In Antimicrobial Peptides from Lactic Acid Bacteria: Diversity, Biosynthesis and Applications (pp. 67-95). Singapore: Springer Nature Singapore. https://doi.org/10.1007/978-981-97-3413-9_3.
Anumudu, C., Hart, A., Miri, T., & Onyeaka, H. (2021). Recent advances in the application of the antimicrobial peptide nisin in the inactivation of spore-forming bacteria in foods. Molecules, 26(18), 5552. https://doi.org/10.3390/molecules26185552.
Mohammad, I., Ansari, M. R., Bari, M. N., Khan, M. S., Kamal, M. A., & Anwar, M. (2025). Enhancing Food Safety: Adapting to Microbial Responses Under Diverse Environmental Stressors. Trends in Ecological and Indoor Environmental Engineering, 3(2), 12–26. https://doi.org/10.62622/TEIEE.025.3.2.12-26
Roque‐Borda, C. A., Primo, L. M. D. G., Medina‐Alarcón, K. P., Campos, I. C., Nascimento, C. D. F., Saraiva, M. M., ... & Albericio, F. (2025). Antimicrobial peptides: a promising alternative to conventional antimicrobials for combating polymicrobial biofilms. Advanced Science, 12(1), 2410893. https://doi.org/10.1002/advs.202410893.
Papadimitriou, K., Alegría, Á., Bron, P. A., De Angelis, M., Gobbetti, M., Kleerebezem, M. & Kok, J. (2016). Stress physiology of lactic acid bacteria. Microbiology and Molecular Biology Reviews, 80(3), 837-890. https://doi.org/10.1128/mmbr.00076-15.
Yap, P. C., MatRahim, N. A., AbuBakar, S., & Lee, H. Y. (2021). Antilisterial potential of lactic acid bacteria in eliminating Listeria monocytogenes in host and ready-to-eat food application. Microbiology Research, 12(1), 234-257. https://doi.org/10.3390/microbiolres12010017.
Mirzapour-Kouhdasht, A., McClements, D. J., Taghizadeh, M. S., Niazi, A., & Garcia-Vaquero, M. (2023). Strategies for oral delivery of bioactive peptides with focus on debittering and masking. npj Science of Food, 7(1), 22. https://doi.org/10.1038/s41538-023-00198-y.
Bhandari, D., Rafiq, S., Gat, Y., Gat, P., Waghmare, R., & Kumar, V. (2020). A review on bioactive peptides: physiological functions, bioavailability and safety. International Journal of Peptide Research and Therapeutics, 26(1), 139-150.
Aluko, R. E. (2018). Food protein-derived peptides: Production, isolation, and purification. In Proteins in food processing (pp. 389-412). Woodhead Publishing. https://doi.org/10.1016/B978-0-08-100722-8.00016-4.
Romdhani, M., Dhaouafi, J., Deracinois, B., Flahaut, C., Nedjar, N., & Balti, R. (2025). Proteomics and bioinformatics guided discovery of microalgal multifunctional peptides for novel nutraceutical applications. Bioprocess and Biosystems Engineering, 1-23. https://doi.org/10.1007/s00449-025-03192-8.
Solieri, L., Sola, L., Vaccalluzzo, A., Randazzo, C. L., Martini, S., & Tagliazucchi, D. (2022). Characterization of cell-envelope proteinases from two Lacticaseibacillus casei strains isolated from Parmigiano Reggiano cheese. Biology, 11(1), 139. https://doi.org/10.3390/biology11010139.
Popoola O. A., Agarry O. O. & Orukotan A. A. (2025). Antimicrobial peptides in non-dairy matrices: Innovations and challenges in biological preservation. In Akinyele B.J., Kayode R. & Akinsemolu A.A. (Eds.), Microbes, Mentorship, and Beyond: A Festschrift in Honour of Professor F.A. Akinyosoye. SustainE. Retrieved from https://sustaine.org/antimicrobial-peptides-in-non-dairy-matrices-innovations-and-challenges-in-biological-preservation/.
Obafemi, Y. D., Oranusi, S. U., Ajanaku, K. O., Akinduti, P. A., Leech, J., & Cotter, P. D. (2022). African fermented foods: overview, emerging benefits, and novel approaches to microbiome profiling. npj Science of Food, 6(1), 15. https://doi.org/10.1038/s41538-022-00130-w.
Benkerroum, N. (2013). Traditional fermented foods of North African countries: technology and food safety challenges with regard to microbiological risks. Comprehensive Reviews in Food Science and Food Safety, 12(1), 54-89. https://doi.org/10.1111/j.1541-4337.2012.00215.x.
Mokoena, M. P., Mutanda, T., & Olaniran, A. O. (2016). Perspectives on the probiotic potential of lactic acid bacteria from African traditional fermented foods and beverages. Food & Nutrition Research, 60(1), 29630. https://doi.org/10.3402/fnr.v60.29630.
Anukam, K. C., & Reid, G. (2009). African traditional fermented foods and probiotics. Journal of medicinal food, 12(6), 1177-1184. https://doi.org/10.1089/jmf.2008.0163.
Ashaolu, T. J., & Adeyeye, S. A. (2024). African functional foods and beverages: a review. Journal of Culinary Science & Technology, 22(1), 142-177. https://doi.org/10.1080/15428052.2022.2034697.
Ismail, T., Layla, A., & Akhtar, S. (2022). Fermented plant protein products. In Plant Protein Foods (pp. 197-222). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-030-91206-2_7.
Nes, I. F., & Holo, H. (2000). Class II antimicrobial peptides from lactic acid bacteria. Peptide Science, 55(1), 50-61. https://doi.org/10.1002/1097-0282(2000)55:1%3C50::AID-BIP50%3E3.0.CO;2-3.
Silpa, S., & Rupachandra, S. (2022). Cyclic peptide production from lactic acid bacteria (LAB) and their diverse applications. Crit. Rev. Food Sci. Nutr, 62(11), 2909-2927. https://doi.org/10.1080/10408398.2020.1860900.
Karnwal, A., & Malik, T. (2024). Exploring the untapped potential of naturally occurring antimicrobial compounds: novel advancements in food preservation for enhanced safety and sustainability. Frontiers in Sustainable Food Systems, 8, 1307210. https://doi.org/10.3389/fsufs.2024.1307210.
Sachdeva, A., Tomar, T., Malik, T., Bains, A., & Karnwal, A. (2025). Exploring probiotics as a sustainable alternative to antimicrobial growth promoters: mechanisms and benefits in animal health. Frontiers in Sustainable Food Systems, 8, 1523678. https://doi.org/10.3389/fsufs.2024.1523678.
Gardoul, M., Rached, B., Mbarki, A., Ajdig, M., Belouad, M., Chouati, T. & El Fahime, E. (2025). Comprehensive whole-genome analysis of Streptococcus infantarius strains from Moroccan farmhouse dairy products: Genomic insights into dairy adaptation, safety, and biotechnological potential. International Journal of Food Microbiology, 111358. https://doi.org/10.1016/j.ijfoodmicro.2025.111358.
Lorente-Torres, B., Castañera, P., Ferrero, H. Á., Fernández-Martínez, S., Adejoh Ocholi, S., Llano-Verdeja, J., ... & Letek, M. (2025). Streamlining Bacillus strain selection against Listeria monocytogenes using a fluorescence-based infection assay integrated into a multi-tiered validation pipeline. Antibiotics, 14(8), 765. https://doi.org/10.3390/antibiotics14080765.
Garcia-Gonzalez, N., Battista, N., Prete, R., & Corsetti, A. (2021). Health-promoting role of Lactiplantibacillus plantarum isolated from fermented foods. Microorganisms, 9(2), 349. https://doi.org/10.3390/microorganisms9020349.
Shaaban, M., Abd El-Rahman, O. A., Al-Qaidi, B., & Ashour, H. M. (2020). Antimicrobial and antibiofilm activities of probiotic Lactobacilli on antibiotic-resistant Proteus mirabilis. Microorganisms, 8(6), 960-1007.
Yang, E., Fan, L., Yan, J., Jiang, Y., Doucette, C., Fillmore, S., & Walker, B. (2018). Influence of culture media, pH and temperature on growth and bacteriocin production of bacteriocinogenic lactic acid bacteria. AMB express, 8, 1-14. https://doi.org/10.1186/s13568-018-0536-0.
Imade, E. E., Omonigho, S. E., Babalola, O. O., & Enagbonma, B. J. (2021). Lactic acid bacterial antimicrobial peptides and their bioactive properties against food-associated antibiotic-resistant bacteria. Annals of Microbiology, 71, 1-14.
Zangeneh, M., Khorrami, S., & Khaleghi, M. (2020). Bacteriostatic activity and partial characterization of the bacteriocin produced by L. plantarum sp. isolated from traditional sourdough. Food Science & Nutrition, 8(11), 6023-6030. https://doi.org/10.1002/fsn3.1890.
R Core Team. (2024). R: A language and environment for statistical computing (Version 4.5.1) (Computer software). R Foundation for Statistical Computing. https://www.R-project.org/.
Wang, J., Yang, X., Peng, Y., Zhang, J., Huang, Y., Zhong, Z. & Peng, G. (2024). Isolation and in vitro investigation on lactic acid bacteria for potential probiotic properties from cat feces. Frontiers in Veterinary Science, 11, 1495745. https://doi.org/10.3389/fvets.2024.1495745.
Zhu, Y., Li, B., Xu, W., Wang, Y., Li, G., Bi, C., & Shao, C. (2025). Association of idealized amphiphiles and protease inhibitors: Conferring antimicrobial peptides with stable antibacterial activity under physiological conditions to combat multidrug-resistant bacteria. Drug Resistance Updates, 79, 101183. https://doi.org/10.1016/j.drup.2024.101183.
Choyam, S., Jain, P. M., & Kammara, R. (2021). Characterization of a potent new-generation antimicrobial peptide of Bacillus. Frontiers in Microbiology, 12, 710741. https://doi.org/10.3389/fmicb.2021.710741.
Gálvez, A., Abriouel, H., López, R. L., & Omar, N. B. (2007). Bacteriocin-based strategies for food biopreservation. International journal of food microbiology, 120(1-2), 51-70. https://doi.org/10.1016/j.ijfoodmicro.2007.06.001.
Parvez, S., Malik, K. A., Ah Kang, S., & Kim, H. Y. (2006). Probiotics and their fermented food products are beneficial for health. Journal of applied microbiology, 100(6), 1171-1185. https://doi.org/10.1111/j.1365-2672.2006.02963.x.
Yang, S. C., Lin, C. H., Sung, C. T., & Fang, J. Y. (2014). Antibacterial activities of bacteriocins: application in foods and pharmaceuticals. Frontiers in microbiology, 5, 241. https://doi.org/10.3389/fmicb.2014.00241.
Cleveland, J., Montville, T. J., Nes, I. F., & Chikindas, M. L. (2001). Bacteriocins: safe, natural antimicrobials for food preservation. International journal of food microbiology, 71(1), 1-20. https://doi.org/10.1016/S0168-1605(01)00560-8.
Sampaio de Oliveira, K. B., Leite, M. L., Rodrigues, G. R., Duque, H. M., da Costa, R. A., Cunha, V. A. & Dias, S. C. (2020). Strategies for recombinant production of antimicrobial peptides with pharmacological potential. Expert review of clinical pharmacology, 13(4), 367-390. https://doi.org/10.1080/17512433.2020.1764347.
Mihaylova-Garnizova, R., Davidova, S., Hodzhev, Y., & Satchanska, G. (2024). Antimicrobial peptides derived from bacteria: classification, sources, and mechanism of action against multidrug-resistant bacteria. International Journal of Molecular Sciences, 25(19), 10788. https://doi.org/10.3390/ijms251910788.
Field, D., Ross, R. P., & Hill, C. (2018). Developing bacteriocins of lactic acid bacteria into next generation biopreservatives. Current Opinion in Food Science, 20, 1-6. https://doi.org/10.1016/j.cofs.2018.02.004.
Chen, X., Bai, H., Mo, W., Zheng, X., Chen, H., Yin, Y. & Peng, H. (2025). Lactic Acid Bacteria Bacteriocins: Safe and Effective Antimicrobial Agents. International Journal of Molecular Sciences, 26(9), 4124. https://doi.org/10.3390/ijms26094124.
Banicod, R. J. S., Tabassum, N., Javaid, A., Kim, Y. M., & Khan, F. (2025). Lactic Acid Bacteria–Derived Secondary Metabolites: Emerging Natural Alternatives for Food Preservation. Probiotics and Antimicrobial Proteins, 1-38. https://doi.org/10.1007/s12602-025-10672-6.
Pang, X., & Lu, Y. (2024). LAB bacteriocin-based strategies for food preservation. In Bio-Based Antimicrobial Agents to Improve Agricultural and Food Safety (pp. 189-220). Bentham Science Publishers. https://doi.org/10.2174/97898152562391240101.
Nishie, M., Nagao, J. I., & Sonomoto, K. (2012). Antibacterial peptides “bacteriocins”: an overview of their diverse characteristics and applications. Biocontrol science, 17(1), 1-16. https://doi.org/10.4265/bio.17.1.
Patel, A., Shah, N., & Verma, D. K. (2017). Lactic Acid Bacteria (Lab) Bacteriocins: An Ecologicaland Sustainable Biopreservativeapproach to Improve the Safety and Shelf Life of Foods. In Microorganisms in Sustainable Agriculture, Food, and the Environment (pp. 197-257). Apple Academic Press. https://doi.org/10.1201/9781315365824.
Pujato, S. A., Mercanti, D. J., Briggiler Marcó, M., Capra, M. L., Quiberoni, A., & Guglielmotti, D. M. (2024). Bacteriocins from lactic acid bacteria: strategies for the bioprotection of dairy foods. Frontiers in Food Science and Technology, 4, 1439891. https://doi.org/10.3389/frfst.2024.1439891.
Sionek, B., Szydłowska, A., Trząskowska, M., & Kołożyn-Krajewska, D. (2024). The impact of physicochemical conditions on lactic acid bacteria survival in food products. Fermentation, 10(6), 298. https://doi.org/10.3390/fermentation10060298.
Aguilar, C., & Klotz, B. (2010). Effect of the temperature on the antagonistic activity of lactic acid bacteria against Escherichia coli and Listeria monocytogenes. Journal of Food Safety, 30(4), 996-1015. https://doi.org/10.1111/j.1745-4565.2010.00257.x.
Qin, H., Gong, S. S., Ge, X. Y., & Zhang, W. G. (2012). The effect of temperature on L-lactic acid production and metabolite distribution of Lactobacillus casei. Preparative Biochemistry and Biotechnology, 42(6), 564-573. https://doi.org/10.1080/10826068.2012.665114.
Hao, F., Fu, N., Ndiaye, H., Woo, M. W., Jeantet, R., & Chen, X. D. (2021). Thermotolerance, survival, and stability of lactic acid bacteria after spray drying as affected by the increase of growth temperature. Food and Bioprocess Technology, 14(1), 120-132. https://doi.org/10.1007/s11947-020-02571-1.
Hernández-Alcántara, A. M., Wacher, C., Llamas, M. G., López, P., & Pérez-Chabela, M. L. (2018). Probiotic properties and stress response of thermotolerant lactic acid bacteria isolated from cooked meat products. Lwt, 91, 249-257. https://doi.org/10.1016/j.lwt.2017.12.063.
Zielińska, D., Krawczyk, M., & Neffe-Skocińska, K. (2025). Thermotolerant Probiotic—The Potential of Improving the Survivability of Beneficial Bacteria. Fermentation, 11(6), 313. https://doi.org/10.3390/fermentation11060313.
Min, B., Yoo, D., Lee, Y., Seo, M., & Kim, H. (2020). Complete genomic analysis of Enterococcus faecium heat-resistant strain developed by two-step adaptation laboratory evolution method. Frontiers in Bioengineering and Biotechnology, 8, 828. https://doi.org/10.3389/fbioe.2020.00828.
Liu, A., Li, X., Pu, B., Ao, X., Zhou, K., He, L., ... & Liu, S. (2017). Use of psychrotolerant lactic acid bacteria (Lactobacillus spp. and Leuconostoc spp.) Isolated from Chinese Traditional Paocai for the Quality Improvement of Paocai Products. Journal of Agricultural and Food Chemistry, 65(12), 2580-2587. https://doi.org/10.1021/acs.jafc.7b00050.
Tanizawa, Y., Tohno, M., Kaminuma, E., Nakamura, Y., & Arita, M. (2015). Complete genome sequence and analysis of Lactobacillus hokkaidonensis LOOC260T, a psychrotrophic lactic acid bacterium isolated from silage. BMC genomics, 16(1), 240. https://doi.org/10.1186/s12864-015-1435-2
Duru, I. C., Ylinen, A., Belanov, S., Pulido, A. A., Paulin, L., & Auvinen, P. (2021). Transcriptomic time-series analysis of cold-and heat-shock response in psychrotrophic lactic acid bacteria. BMC genomics, 22(1), 28. https://doi.org/10.1186/s12864-020-07338-8
Heng, Y. C., Silvaraju, S., Lee, J. K. Y., & Kittelmann, S. (2023). Lactiplantibacillus brownii sp. nov., a novel psychrotolerant species isolated from sauerkraut. International Journal of Systematic and Evolutionary Microbiology, 73(12), 006194. https://doi.org/10.1099/ijsem.0.006194.
Van Bokhorst-van de Veen, H., Bongers, R. S., Wels, M., Bron, P. A., & Kleerebezem, M. (2013). Transcriptome signatures of class I and III stress response deregulation in Lactobacillus plantarum reveal pleiotropic adaptation. Microbial Cell Factories, 12(1), 112. https://doi.org/10.1186/1475-2859-12-112.
Tenea, G. N. (2022). Decoding the gene variants of two native probiotic Lactiplantibacillus plantarum strains through whole-genome resequencing: Insights into bacterial adaptability to stressors and antimicrobial strength. Genes, 13(3), 443. https://doi.org/10.3390/genes13030443.
Papadimitriou, K., Alegría, Á., Bron, P. A., De Angelis, M., Gobbetti, M., Kleerebezem, M. & Kok, J. (2016). Stress physiology of lactic acid bacteria. Microbiology and Molecular Biology Reviews, 80(3), 837-890. https://doi.org/10.1128/mmbr.00076-15.
Papadopoulou, E., de Evgrafov, M. C. R., Kalea, A., Tsapekos, P., & Angelidaki, I. (2023). Adaptive laboratory evolution to hypersaline conditions of lactic acid bacteria isolated from seaweed. New Biotechnology, 75, 21-30. https://doi.org/10.1016/j.nbt.2023.03.001.
Khushboo, Karnwal, A., & Malik, T. (2023). Characterization and selection of probiotic lactic acid bacteria from different dietary sources for development of functional foods. Frontiers in microbiology, 14, 1170725. https://doi.org/10.3389/fmicb.2023.1170725.
Nami, Y., Panahi, B., Jalaly, H. M., Rostampour, M., & Hejazi, M. A. (2025). Probiotic characterization of LAB isolated from sourdough and different traditional dairy products using biochemical, molecular and computational approaches. Probiotics and Antimicrobial Proteins, 17(3), 1014-1037. https://doi.org/10.1007/s12602-024-10234-2.
Ndiaye, A., Fliss, I., & Filteau, M. (2024). High-throughput characterization of the effect of sodium chloride and potassium chloride on 31 lactic acid bacteria and their co-cultures. Frontiers in Microbiology, 15, 1328416. https://doi.org/10.3389/fmicb.2024.1328416.
Wu, D., Dai, M., Shi, Y., Zhou, Q., Li, P., & Gu, Q. (2022). Purification and characterization of bacteriocin produced by a strain of Lacticaseibacillus rhamnosus ZFM216. Frontiers in Microbiology, 13, 1050807. https://doi.org/10.3389/fmicb.2022.1050807.
Ishikawa, M., Nakajima, K., Yanagi, M., Yamamoto, Y., & Yamasato, K. (2003). Marinilactibacillus psychrotolerans gen. nov., sp. nov., a halophilic and alkaliphilic marine lactic acid bacterium isolated from marine organisms in temperate and subtropical areas of Japan. International journal of systematic and evolutionary microbiology, 53(3), 711-720. https://doi.org/10.1099/ijs.0.02446-0.
Sionek, B., Szydłowska, A., Trząskowska, M., & Kołożyn-Krajewska, D. (2024). The impact of physicochemical conditions on lactic acid bacteria survival in food products. Fermentation, 10(6), 298. https://doi.org/10.3390/fermentation10060298.
Nyanga-Koumou, A. P., Ouoba, L. I. I., Kobawila, S. C., & Louembe, D. (2012). Response mechanisms of lactic acid bacteria to alkaline environments: a review. Critical reviews in microbiology, 38(3), 185-190. https://doi.org/10.3109/1040841X.2011.640978.
Malik, E., Dennison, S. R., Harris, F., & Phoenix, D. A. (2016). pH dependent antimicrobial peptides and proteins, their mechanisms of action and potential as therapeutic agents. Pharmaceuticals, 9(4), 67. https://doi.org/10.3390/ph9040067.
Sanchez-Reinoso, Z., Cournoyer, A., Thibodeau, J., Said, L. B., Fliss, I., Bazinet, L., & Mikhaylin, S. (2021). Effect of pH on the antimicrobial activity and peptide population of pepsin hydrolysates derived from bovine and porcine hemoglobins. ACS Food Science & Technology, 1(9), 1687-1701. https://doi.org/10.1021/acsfoodscitech.1c00141
Pham, H. T., Shi, W., Xiang, Y., Foo, S. Y., Plan, M. R., Courtin, P. & Turner, M. S. (2021). Cyclic di-AMP oversight of counter-ion osmolyte pools impacts intrinsic cefuroxime resistance in Lactococcus lactis. MBio, 12(2), 10-1128. https://doi.org/10.1128/mbio.00324-21.
Bhowmick, S., Shenouda, M. L., & Tschowri, N. (2023). Osmotic stress responses and the biology of the second messenger c-di-AMP in Streptomyces. Microlife, 4, uqad020. https://doi.org/10.1093/femsml/uqad020.
Pandit, G., Sarkar, T., Debnath, S., Satpati, P., & Chatterjee, S. (2022). Delineating the mechanism of action of a protease resistant and salt tolerant synthetic antimicrobial peptide against Pseudomonas aeruginosa. ACS omega, 7(18), 15951-15968. https://doi.org/10.1021/acsomega.2c01089.
Azzahra, R. N., Putri, F. D., Wulandari, A. P., Lei, J., & Syaputri, Y. (2025). Plantaricin: Production Dynamics, Functional Applications, and Challenges in Food Biopreservation. Journal of Food Processing and Preservation, 2025(1), 6215738. https://doi.org/10.1155/jfpp/6215738.
Choudhary, S., Shanu, K., & Devi, S. (2024). Application of AMPs in the Food and Beverage Industry. In Antimicrobial Peptides from Lactic Acid Bacteria: Diversity, Biosynthesis and Applications (pp. 247-281). Singapore: Springer Nature Singapore. https://doi.org/10.1007/978-981-97-3413-9_10.
Mercer, D. K., Torres, M. D., Duay, S. S., Lovie, E., Simpson, L., von Köckritz-Blickwede, M., ... & Angeles-Boza, A. M. (2020). Antimicrobial susceptibility testing of antimicrobial peptides to better predict efficacy. Frontiers in cellular and infection microbiology, 10, 326. https://doi.org/10.3389/fcimb.2020.00326.
Snyder, A. B., & Worobo, R. W. (2014). Chemical and genetic characterization of bacteriocins: antimicrobial peptides for food safety. Journal of the Science of Food and Agriculture, 94(1), 28-44. https://doi.org/10.1002/jsfa.6293.
Singh, S., Jha, B., Tiwari, P., Joshi, V. G., Mishra, A., & Malik, Y. S. (2024). Recent approaches in the application of antimicrobial peptides in food preservation. World Journal of Microbiology and Biotechnology, 40(10), 315. https://doi.org/10.1007/s11274-024-04126-4.

No competing interests reported.

Download PDF

Journal Publication

published 25 Nov, 2025

Read the published version in BMC Biotechnology →

Editorial decision: Revision requested
15 Oct, 2025
Reviews received at journal
05 Oct, 2025
Reviewers agreed at journal
28 Sep, 2025
Reviews received at journal
25 Sep, 2025
Reviewers agreed at journal
16 Sep, 2025
Reviewers agreed at journal
09 Sep, 2025
Reviewers invited by journal
09 Sep, 2025
Editor invited by journal
09 Sep, 2025
Editor assigned by journal
05 Sep, 2025
Submission checks completed at journal
05 Sep, 2025
First submitted to journal
05 Sep, 2025

You are reading this latest preprint version

Protease Sensitivity and Stress Adaptation of Bioactive Peptide-Producing Lactic Acid Bacteria: Functional Implications for Food Biopreservation

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Methods

Bacterial strains and culture conditions

Physiological stress assays

Antimicrobial activity assays

Preparation of cell-free supernatants from 13 AMP-producing LAB strains

Agar well diffusion assay

Protease sensitivity tests

Statistical analysis

Results

Antimicrobial peptide-producing bacterial strains

Effect of physiological stress on AMP-producing LAB

Growth of AMP-producing LAB strains at different temperatures

Growth of AMP-producing LAB under different NaCl concentrations

Growth of AMP-producing LAB strains at different pH

Protease sensitivity of AMPs

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1