Anti-listerial activity of active high-methoxyl pectin films incorporated with Latilactobacillus curvatus CRL705 bacteriocins: effectiveness assessment in ham

Listeria monocytogenes is a foodborne pathogen able to survive, proliferate at refrigeration temperatures and contaminate a wide variety of foods, particularly chilled meat and ready-to-eat (RTE) meat products. One of the major challenges for the food industry in preventing the proliferation of spoilage and pathogen microorganisms lies in food packaging innovation. In this study, edible films were developed to control Listeria monocytogenes in cooked ham. An active film with antimicrobial properties was formulated from high-methoxyl pectin (HMP) and lactocin AL705 (AL705), a bacteriocin produced by Latilactobacillus (Lat.) curvatus CRL705. The optical, mechanical, and antimicrobial properties of the edible films were measured to determine the effects of AL705 on the film matrix. The minimum inhibitory concentration (MIC) of the bacteriocin used for film activation was 1.25 mg/mL. The incorporation of AL705 into the films did not significantly affect (p ≥ 0.05) moisture content, color parameters (L, a, DE*), elongation at break, or tensile strength compared with control films (HMP/Control). However, HMP/AL705 films exhibited lower water vapor permeability (p ≤ 0.05) and reduced transparency. The incorporation of AL705 did not significantly alter peak intensity or generate new peaks in the FTIR spectra. SEM micrographs revealed surface modifications in films containing bacteriocin AL705 compared with the control films. In edible films activated with AL705 at 4 °C, the growth of L. monocytogenes was completely inhibited in cooked ham for 7 days. Although the Listeria load increased toward day 14, it remained satisfactorily controlled in samples treated with HMP/AL705 films compared with samples treated with HMP/Control films. A similar trend was observed at 10 °C, which was used as the abuse temperature. Therefore, HMP films incorporated with lactocin AL705 may represent a promising alternative for functional food packaging aimed at improving food preservation.

Listeria monocytogenes is a foodborne pathogen responsible for caused listeriorisis, a disease that can manifest as gastroenteritis, abortion, or sepsis depending largely on the host’s immune system condition (Schlech, 2019). The ability of certain strains to tolerate biocides ensures their persistence in production environments, which poses a threat to food safety and public health (Mohamed et al., 2018). L. monocytogenes contaminates a wide variety of foods, particularly chilled meat and ready-to-eat (RTE) meat products, because it can survive and proliferate at refrigeration temperatures (Jiang and Xiong, 2015). In this context, one of the major challenges for the food industry in preventing the proliferation of spoilage and pathogen microorganisms lies in food packaging innovation. Antimicrobial packaging is an emerging technology that combines packaging materials with antimicrobial agents to preserve food quality and ensure safety (Sohail et al., 2018). Among natural agents, bacteriocins, which are ribosomally synthesized peptides produced by certain lactic acid bacteria, are promising candidates for bioactive packaging applications (Motelica et al., 2020). Research has increasingly focused on active films incorporating bacteriocins with anti-listerial activity (Gumienna and Górna, 2021). The best-characterized bacteriocins for such applications include nisin (Carrión et al., 2023; Khan et al., 2014; Maresca and Mauriello, 2022), pediocin PA-1/AcH (Espitia et al., 2013; Woraprayote et al., 2013), and enterocin (Aymerich et al., 2022; Ortega Blázquez et al., 2018). Latilactobacillus (Lat.) curvatus CRL705, isolated from traditional Argentinean fermented sausages, has been shown to harbor bacteriocin-encoding genes (Hebert et al., 2012). Previous studies have demonstrated the antagonist effect of these bacteriocins against lactic acid bacteria and Listeria species (Castellano and Vignolo, 2006). In particular, lactocin 705 (class IIb) and lactocin AL705 (class IIa), produced by Lat. curvatus CRL705, were adsorbed onto linear low-density polyethylene (LLDPE) films and exhibited strong inhibitory effects against Listeria (Blanco Massani et al., 2008). Furthermore, wheat gluten films enriched with Lat. curvatus CRL705 bacteriocins effectively prevented Listeria growth in wieners stored at 5 °C for 45 days (Blanco Massani et al., 2014). However, only nisin and pediocin PA-1/AcH are currently approved as food additives; therefore, further studies evaluating new bacteriocins in different matrices are necessary to support their application and advance the development of functional antimicrobial packaging systems. In parallel, there is increasing interest in eco-friendly active packaging made from bio-based polymers, such as cellulose, pectin, starch, and polylactic acid (PLA), due to their biodegradability and functional versatility (Gumienna and Górna, 2021). Among these materials, pectin is particularly valued for its techno-functional properties, such as biodegradability, biocompatibility, antimicrobial activity, physical properties, non-toxicity, and low cost, which make it suitable for film formation (Butler et al., 2023; Pasini Cabello et al., 2015). Based on this, the purpose of our study was to characterize a high-methoxyl pectin (HMP)-based packaging film containing the bacteriocins produced by Lat. curvatus CRL705 and to evaluate its effectiveness against L. monocytogenes in cooked ham, with potential application in ready-to-eat (RTE) meat products.

Strains and culture conditions

Listeria (L.) monocytogenes FBUNT (Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Argentina) was routinely cultivated in Triptein Soya Broth (TSB; BBL, Cockeysville, MD, USA) supplemented with 0.5% (w/v) yeast extract (YE) for 16 h at 30 °C. Lat. curvatus CRL705, a producer of lactocin 705 (two-component bacteriocin) and lactocin AL705 (anti-listerial bacteriocin), was grown in MRS broth at 30 °C (Castellano and Vignolo, 2006). Culture stocks were maintained and stored at -20 °C in the corresponding liquid media supplemented with 15% (v/v) glycerol. Unless otherwise stated, all media used were supplied by Britania (Buenos Aires, Argentina).

Bacteriocin purification and minimum inhibitory concentration

One liter of an overnight culture of Lat. curvatus CRL705 grown in MRS broth was centrifuged (10,000 x g, 10 min at 4 °C; Avanti J-25I centrifuge, Beckman, USA) to remove bacterial cells. The bacteriocins (lactocin 705 and lactocin AL705) were precipitated from the supernatants with 60% saturated ammonium sulfate (Cicarelli-Reagents S.A, Santa Fé, Argentina) under stirring for 30 min at 4 °C. The extract was then centrifuged (11,000 x g, 10 min at 4 °C), and the resulting pellet was solubilized in 20 mL of 20 mM phosphate buffer (pH 6.5). Finally, the semi-purified bacteriocin solution was lyophilized and stored at -20 °C until use. The MIC of lactocin AL705 against L. monocytogenes FBUNT was determined by the broth microdilution method following the Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute (CLSI), 2017). Briefly, two-fold dilutions of lactocin AL705 were prepared using Mueller-Hinton broth (MHB) in 96-well microplates. A Listeria inoculum standardized to 0.5 McFarland was added to obtain a total volume of 200 μL per well. After 20 h of incubation at 30 °C, the absorbance at 600 nm (OD600) was measured in each well using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The MIC was defined as the lowest concentration of lactocin AL705 that resulted in at least a 95% reduction in pathogen growth. Growth inhibition percentages were calculated as described by Melian et al. (2019).

Preparation of edible film containing bacteriocin

Films were prepared from apple high-methoxyl pectin (HMP) supplied by Grupo Saporiti S.A (Buenos Aires, Argentina). The films were prepared according to the protocol described by Matta and Bertola (2020) with minor modifications. To prepare film-forming solutions (FFS), 2.8% (w/v) HMP was dispersed in distilled water at 50 °C for 2 h under constant magnetic stirring. Then, glycerol (22% v/v) was added to the HMP solution as a plasticizer. After complete dissolution of HMP, lactocin AL705 was incorporated at different concentrations (0.62, 1.25, and 2.50 mg/mL). The pH was adjusted to 3.0 using a 1 N HCl solution. Approximately 25 g of FFS, with and without lactocin AL705, was cast onto Petri dishes (9 cm diameter) and dried at 30 °C in an oven until a constant weight was reached. The films were designated as HMP/Control (without bacteriocin) and HMP/AL705 (anti-listerial active film). Prior to testing, the films were conditioned at 21 °C ± 2 and 65% relative humidity (RH) for 48 h.

Antimicrobial activity of active films

Control and AL705-activated HMP films were placed on semi-solid TSB agar supplemented with YE plates inoculated with 10 6 CFU/mL of the indicator strain L. monocytogenes FBUNT. After overnight incubation at 30 °C, the antimicrobial activity was evidenced by an inhibition zone around the films.

Characterization of HMP/Control and HMP/AL705 films

Film thickness

Film thickness was measured using a hand-held micrometer (CM-8826FN, BYQTEC, China). Thickness was determined as the arithmetic mean of twelve random measurements (from the center and areas near the perimeter) of each film. The average thickness was used for calculating opacity, water vapor permeability, and mechanical properties.

Color parameter measurement

Color parameters L *, a *, and b * (brightness, redness, and yellowness, respectively), C * (chromaticity), and YI (yellowness index) were determined using a Konica Minolta CR-400 colorimeter (Osaka, Japan) based on the CIE color system. An average color value for each sample was determined from five sides of the films. The CIE-Lab scale was used with a D65 light source and a 10° observation angle. A white plate (L * = 91.36, a * = -0.35, and b * = 2.94) was used as a standard to calculate the color difference (ΔE *) of the films against a white background, which was calculated using the following (Equation 1):

Where △L *, △a *, and △b * represent the differences between the color parameters of the sample and the film background. The parameters C* and YI were calculated following (Equations 2, 3) respectively.

and

(Younis and Zhao, 2019)

Optical characterization of HMP films

Light transmission and opacity of the films were determined using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Ultraviolet (UV) barrier properties of the films were measured at wavelengths ranging from 200 to 400 nm. The percentage of transmittance was determined at 600 nm, and the opacity value was calculated using the following (Equation 4):

Where A 600 is the value of absorbance at 600 nm, and T is the film thickness (mm).

Contact angle measurements

The surface hydrophobicity of HMP/AL705 films was determined by the sessile drop method (Karbowiak et al., 2006), using a contact angle meter (DSA100, KRÜSS, Hamburg, Germany) equipped with image analysis software (Krüss Advance, Hamburg, Germany, version 1.4.1.2).

Moisture content and water vapor permeability

The moisture content of the films was determined by measuring their weight loss upon drying in an oven at 105 °C until constant weight was achieved. Moisture content was calculated as the weight difference between the films before and after drying. The water vapor permeability (WVP) was determined using a modified gravimetric method, ASTM E96 (Tavera Quiroz et al., 2013). Each film sample was placed on top of hermetically sealed acrylic cups containing anhydrous silica desiccant (0% RH) and stored in a desiccator maintained at 75% RH and 25 °C. A driving force (ΔP) of 1753.55 Pa was used, corresponding to a 75% RH gradient across the film. When steady-state conditions were reached, the permeation cells were weighed at 1 h intervals for approximately 7 h. WVP was calculated as follows (Equation 5):

Where WVTR (water vapor transmission rate) was calculated from the slope of the straight line (g/s) divided by the cell area. S is the saturation vapor pressure of water (Pa), R1 is the RH in the desiccator, R2 is the RH in the permeation cell, and d is the film thickness. All measurements were performed in triplicate, and mean WVP values were reported.

Determination of tensile strength and elongation at break

Tensile strength (TS) and elongation at break (EB) in the control and active films were evaluated using a CT3 Texture Analyzer (Brookfield, WI, USA) according to the ASTM D882-02. The films were cut into strips (70 mm × 10 mm), and the results were averaged from five parallel measurements. The initial distance between tensile grips was set at 50 mm. TS was calculated using Equation (6), followed by the calculation of EB using Equation (7).

TS = FM/A (6) where TS is tensile strength (MPa), FM is the maximum force at the time of film break (N), and A is the cross-sectional area (m 2).

EB = (DR/DI) × 100 (7) where EB represents elongation (%), DR is the distance at the moment of rupture (cm), and DI is the initial separation distance (cm).

Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectra of the different films were obtained using an Agilent Cary 630 FTIR Spectrometer (Agilent Technologies, Inc., Santa Clara, California, USA). Spectra were recorded over the wavenumber range of 4000–400 cm - ¹ using 64 scans at a resolution of 4 cm - ¹.

Microstructure analysis of films by SEM

The microstructure of the film surfaces was visualized using a scanning electron microscope (SEM, Hitachi SU 1510, Irvine, CA, USA). Control films and films containing 1.25 mg/mL lactocin AL705 were cryo-fractured by immersion in liquid nitrogen. Prior to visualization, the film samples were mounted on brass stubs and sputter-coated with gold to make the samples conductive. The samples were photographed at an accelerating voltage of 3 kV. The images were taken at magnifications of 2000× and 50000×.

Treatment of cooked ham samples with HMP-based film

Cooked ham was cut into slices measuring approximately 2.5 × 2.5 × 0.3 cm and sterilized under UV light for 30 min. The samples were inoculated on both sides with a diluted overnight culture (0.1 mL; 10 4 CFU/mL) of L. monocytogenes using a sterile pipette. The inoculated samples were dried for 10 min under a laminar flow hood, flipped over, and inoculated on the opposite side. The inoculated ham pieces were wrapped in the active film containing lactocin AL705 (1.25 mg/mL). Similarly, film-coated ham samples without bacteriocins and ham pieces without film were used as controls. All samples were placed in bags (Cryovac, Buenos Aires, Argentina) with an oxygen diffusion coefficient of 6/14 cm 3 m -2 atm -1– 24 h -1 at 25 °C and 75% relative humidity. The packages were sealed to a final vacuum of 98% using a Turbovac 320 ST vacuum packaging machine (HFE Vacuum Systems, Hertogenbosh, Netherlands). One set of samples was incubated at 4 °C for 14 days, while another set was stored at 10 °C for the same period. These conditions were chosen considering local regulations Administración Nacional de Medicamentos, Alimentos y Tecnología Médica (ANMAT); Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), 1968) with the aim of simulating temperature changes during storage and handling in commercial and domestic environments. Triplicate ham samples from each treatment and sampling interval (0, 7, and 14 days) were analyzed for L. monocytogenes counts. The sample were mixed (1:10) with sterile saline solution (NaCl 0.85% w/v) and homogenized in a Stomacher Lab-blender Model 400 (Seward Laboratory, London, UK) for 1 min. Serial 10-fold dilutions were then prepared using sterile peptone water (0.1% w/v) and plated on PALCAM selective media (Difco Laboratories, Inc., Detroit, MI, USA). The plates were incubated at 30 °C for 48 h, and colony counts were expressed as log of colony-forming units per gram (log CFU/g). The pH was also measured using a Metrohn 692 pH/Ion Meter with temperature compensation (Metrohn SA, Herisau, Switzerland). The equipment was calibrated using 4.0 and 7.0 buffer standards, and all measurements were performed during the same session.

Statistical analysis

All experiments were performed in triplicate using three independent batches. Means and standard deviations were calculated. Significant differences (p ≤ 0.05) among means were determined by analysis of variance (ANOVA), followed by Tukey’s test, using the statistical program INFOSTAT software version 2020 (InfoStat Group, Córdoba, Argentina).

Anti-listerial activity of bioactive films

The MIC of lactocin AL705 against L. monocytogenes FBUNT was 1.25 mg/mL, defined as the lowest concentration achieving ≥95% growth inhibition Concentrations corresponding to 1/2 MIC, MIC, and 2 MIC were selected to make the films.

Lactocin AL705 was incorporated into HMP-based films at concentrations of 0.62, 1.25, and 2.50 mg/mL to evaluate its antimicrobial efficacy against L. monocytogenes. Films containing 1.25 and 2.50 mg/mL of lactocin AL705 demonstrated clear inhibitory activity, while no inhibition was observed at 0.62 mg/mL (Supplementary Figure 1). Therefore, the MIC was established at 1.25 mg/mL, defined as the lowest concentration capable of completely inhibiting visible bacterial growth around the film.

Characterization of HMP/Control and HMP/AL705 films

The addition of active compounds can modify the physicochemical and mechanical properties of packaging films. Therefore, three different concentrations of lactocin AL705 (1/2 MIC, MIC, and 2 MIC) were evaluated in this study.

Optical properties

The color parameters (L *, a *, b *, Δ E *, C *, and YI) of the control and lactocin AL705-treated films are shown in Table 1. The parameter L * corresponds to luminosity (0=black and 100=white), whereas a * and b * correspond to color parameters whose distribution is green (-)/red (+) and blue (-)/yellow (+), respectively. The incorporation of lactocin AL705 at different concentrations (0.62, 1.25, and 2.50 mg/mL) did not significantly alter L * (lightness) values (79.42, 77.88, 78.56 respectively) compared with those of the HMP/Control films (74.36) (p ≥ 0.05). The a * (greenness) values obtained for the HMP/Control films (-0.34) and the HMP/AL705 films containing 0.62 mg/mL (-0.44) and 1.25 mg/mL (-0,36) did not show significant differences. However, films with a higher concentration of bacteriocin (-0.24) did show significant differences with the control films. However, increasing concentrations of the bacteriocin resulted in significantly higher b * values (yellow component), chromaticity (C *), and yellowness index (YI) (p ≤ 0.05). Δ E * values remained statistically similar across all formulations. Visually, the treated films appeared more opaque and slightly yellowish than the controls. The UV absorbance spectra (Figure 1) revealed increased absorption in the UVC range (200–280 nm) for AL705-incorporated films, with no notable absorption in the 300–400 nm range.

Water contact angle, moisture content, permeability, and mechanical properties

The surface properties of the films were examined using water contact angle (WCA, θ) measurements. The HMP/Control films used in this study exhibited an initial contact angle of 38°, indicating a moderately hydrophilic character due to the presence of carboxyl groups (Figure 2). The WCA analysis showed slightly lower values for the AL705-containing films compared with the controls; however, these differences were not statistically significant (p ≥ 0.05).

Table 2 presents the moisture content, WVP values, and mechanical properties of all film formulations. The HMP/Control films exhibited a moisture content of approximately 9%, while the HMP/AL705 films did not show changes in this parameter compared with the control films. However, WVP decreased progressively with increasing bacteriocin concentration, with the lowest WVP value (3.24 ×10– 10 g m -1 s -1 Pa -1) observed in films containing 2.5 mg/mL of AL705.

Regarding the mechanical properties of the films, the TS and EB values are shown in Table 2. The HMP/Control films showed TS and EB values of 0.13 ± 0.01 MPa and 15.11 ± 1.52%, respectively. The incorporation of lactocin AL705 into the films did not significantly affect their mechanical properties.

Structural analysis of HMP films

The FTIR spectra of the HMP/Control and HMP/AL705 films are shown in Figure 3. The FTIR spectra of the bioactive films indicated that the chemical structure of the films remained unchanged after bacteriocin addition. This antimicrobial compound did not produce significant differences either in the intensity or in the appearance of new peaks. Both HMP/control and HMP/AL705 films exhibited major absorption bands in the 3100–2900 cm - ¹ region, corresponding to the stretching and bending vibrations of –CH bonds present in the pectin polymer chains. Characteristic peaks associated with pectin functional groups were observed at 1740 cm - ¹ (aldehyde stretching) and 1645 cm - ¹ (amide I, symmetric stretching of C=O). Additional bands appeared at 1320–1350 cm - ¹ (C–O stretching) and at 1220–1250 cm - ¹ (asymmetric C–O–C stretching), indicating the presence of abundant methoxyl groups. The strong absorption band at 1015 cm - ¹ corresponded to symmetric C–O–C stretching vibrations of the saccharide backbone, confirming the high degree of esterification and the predominance of high-methoxyl pectin in the studied films. Bands observed between 900 and 400 cm - ¹ were associated with vibrations of sugars in the pyranose form.

On the other hand, the morphology of the films was observed by SEM (Figure 4). The micrographs revealed that the HMP/Control films displayed a uniform and compact surface, whereas the incorporation of lactocin AL705 (1.25 mg/mL) induced slight morphological changes, resulting in a rougher surface without evidence of aggregation.

Antimicrobial activity of HMP-based films on cooked ham samples

The inhibitory effect of active HMP films against L. monocytogenes FBUNT inoculated onto cooked ham slices and incubated at 4 and 10 °C for 14 days was evaluated (Figure 5). Viable counts of L. monocytogenes in ham coated with HMP/AL705 films were not detectable at 0 and 7 days of incubation at either temperature. Although an increase in Listeria load was observed after day 7 (~4.2 ± 0.18 log CFU/g at both 4 °C and 10 °C), the active films effectively controlled bacterial growth throughout the storage period compared with the control samples. Cooked ham samples without films or coated with the control HMP films exhibited an initial cell count of ∼ 4.33 ± 0.15 log CFU/g, reaching values of ∼ 9.32 ± 0.04 log CFU/g at the end of storage period for both temperatures.

The inhibitory effect of the HMP films active against L. monocytogenes FBUNT inoculated onto slices of cooked ham and incubated at 4 and 10 °C for 14 days was evaluated (Figure 5). No viable L. monocytogenes counts were detected in the ham coated with HMP/AL705 films at 0 and 7 days of incubation at either temperature. In contrast, ham samples without film or coated with HMP film showed an initial count greater than 4 log CFU/g at both 4 °C and 10 °C. After 7 days of incubation, an increase in Listeria load was observed at both temperatures. The samples without film showed a count of 6.40 ± 0.12 log CFU/g at 4 °C and 7.49 ± 0.08 log CFU/g at 10 °C. Similar values ​​were obtained in the samples coated with the control film (6.29 ± 0.27 log CFU/g and 6.91 ± 0.68 log CFU/g, for 4 and 10 °C, respectively). At 14 days of incubation, in the control samples, Listeria counts greater than 9 logarithmic units were recorded. In contrast, in the samples coated with the active film, reductions of ~5 logarithmic units in the pathogen count were observed.

In the current study, the incorporation of lactocin AL705 into HMP-based films resulted in a multifunctional material with promising properties for active food packaging. The antimicrobial activity of films containing lactocin AL705, with a MIC value of 1.25 mg/mL, was consistent with previous findings, indicating the potency of this bacteriocin when incorporated into biopolymer matrices. Blanco Massani et al. (2008) reported a similar MIC for lactocin AL705 embedded in polyethylene-based films against L. innocua 7, used as a non-pathogenic surrogate for L. monocytogenes. In comparison, Yang et al. (2020) demonstrated that cellulose nanofibril (CNF) films containing 1920 mg/L of nisin completely inhibited L. monocytogenes during seven days of storage at 4 °C in ready-to-eat ham. However, composite films treated with chitosan and divergicin M35 at 0,125 mg/mL effectively suppressed L. monocytogenes LSD532 on cold smoked salmon (Benabbou et al., 2018). Variations in film composition, bacteriocin type, and food matrix may explain these differences in antimicrobial effectiveness. In addition to antimicrobial efficacy, the physicochemical and mechanical properties of packaging materials are critical for their practical application in the food industry.

Therefore, in this study, the effect of incorporating lactocin AL705 into HMP-based films was also evaluated in terms of their physicochemical and mechanical behavior. Regarding the chromaticity parameter L (luminosity), the HMP films exhibited high L values. Other authors have also reported high L values ​​for pectin films, reflecting their nearly colorless nature and high brightness (Younis et al., 2020; Shankar et al., 2016). The maintenance of lightness (L *) despite bacteriocin addition suggests that the films remain visually white, a desirable attribute for food packaging. A similar trend for biodegradable gelatin-based films containing a nisin-EDTA mixture was reported by Abarca et al. (2022). As for the chromaticity parameter b, bacteriocin addition induced an increase in b, C, and YI values with increasing bacteriocin concentration. In contrast, Younis et al. (2020) reported a decrease in the chromaticity parameters b, C, and YI after the incorporation of fibers into pectin films. The slight increase in yellowness at higher bacteriocin concentrations may be attributed to the intrinsic coloration of lactocin AL705. Opacity also increased significantly at the highest bacteriocin concentration, indicating reduced transparency. Comparable behavior has been reported in fish gelatin films containing mango shell extract, where increasing extract concentration led to lower transparency (Adilah et al., 2018). Opacity of pectin films was also affected by the addition of small sugarcane fibers (Younis et al., 2020). In contrast, Haghighi et al. (2020) reported transparent chitosan–polyvinyl alcohol films regardless of the presence of ethyl lauroyl arginate, highlighting that the effect of antimicrobial incorporation on optical properties depends on both the active compound and the polymer matrix.

In addition, the UV-barrier property is very important for evaluating food packaging films. Good barrier properties help prevent oxidation of foods and, consequently, reduce nutrient loss, discoloration, and unpleasant flavors. The films obtained in this study showed major absorbance in UVC regions (200–280 nm), evidenced by the presence of peaks near 200 nm and a broad peak in the 275 nm region. In contrast, no notable absorption was detected in the near-UV range (300–400 nm). These results align with findings by Younis et al. (2020), who reported UV spectra of pectin-based films exhibiting peaks in the mid-UV range (200–300 nm). Similarly, Shankar et al. (2016) observed strong UV-blocking properties in pectin films, indicated by pronounced absorption in the UV region. The increase in absorbance observed in the present study may be attributed to the presence of specific compounds within the film that absorb short-wavelength UV radiation, which may also contribute to increased opacity. Comparable UV-barrier characteristics have been reported in other biopolymeric films, such as those based on carboxymethyl cellulose, alginate, and k-carrageenan/konjac glucomannan blends (Duan et al., 2021; Achachlouei and Zahedi, 2018; Tang et al., 2018). Therefore, the incorporation of lactocin AL705 appears to enhance the UV-protective properties of the films, which is a desirable feature for preventing oxidative deterioration in packaged food products.

The results for WVP indicated a significant difference between films with and without the bacteriocin. The incorporation of lactocin AL705 resulted in a noticeable reduction in WVP values, enhancing the suitability of the films for applications requiring strong water vapor barrier properties. This effect can be attributed to the presence of lactocin AL705, since both control and bioactive films contained the same concentration of the hydrophilic plasticizer. This reduction may be explained by the dispersion of lactocin AL705 particles within the pectin–glycerol matrix, which promotes intermolecular interactions that limit the penetration of water molecules. In contrast, other authors reported that the addition of mango peel extract did not induce significant changes in the WVP of gelatin films, although a decreasing trend was observed (Adilah et al., 2018). On the other hand, bacteriocin incorporation did not significantly affect the mechanical properties of the films. Therefore, lactocin AL705 was able to reduce the films’ WVP without altering TS or EB at any of the tested concentrations. In contrast, the addition of nisin at concentrations above 1920 mg/L led to a significant decrease in the TS of cellulose nanofibril films, due to the disruption of the CNF network integrity, whereas lower concentrations (620 and 1280 mg/L) did not produce this effect. Similarly to our findings, divergicin M35 reduced the WVP of chitosan-based films without affecting their mechanical strength (Benabbou et al., 2020). Conversely, Rivera-Hernández et al. (2021) reported that nisin decreased both TS and EB of pectin-gellan films without causing significant changes in WVP.

In this study, FTIR analysis was employed to evaluate the impact of lactocin AL705 incorporation on intermolecular interactions in HMP films. The results revealed that the chemical structure of the bioactive films remained unchanged after bacteriocin addition. The antimicrobial compound did not cause noticeable variations in peak intensity or the appearance of new absorption bands. This stability can be attributed to the relatively low incorporation level of the bacteriocin compared with the biopolymer matrix and plasticizer used. Similar findings have been reported for bioactive pectin films incorporating silver nanoparticles (Shankar et al., 2016) and essential oils (Nisar et al., 2018), where no significant structural changes were detected. Likewise, Yang et al. (2020) observed that direct incorporation of nisin into cellulose nanofibril (CNF) films did not promote any chemical interactions between the two components. Both HMP/Control and HMP/AL705 films displayed characteristic peaks corresponding to stretching vibrations of methylene and methyl groups, consistent with previous reports for pectin-based materials (Shankar et al., 2016; Pasini Cabello et al., 2015). In agreement with Younis et al. (2020), minor absorption bands related to sugar residues were observed in the 1400–1200 cm - ¹ and 900–400 cm - ¹ regions. Furthermore, typical carboxyl and carbonyl group vibrations appeared between 1650 and 1400 cm - ¹, consistent with previously published FTIR spectra of pectin films (Gupta et al., 2014; Jridi et al., 2020). The high degree of esterification of the pectin was confirmed by the pronounced peaks at 1200–1250 cm - ¹, corresponding to methoxyl group vibrations. Zanella and Pereira Taranto (2015) reported comparable absorption patterns between 1200 and 1100 cm - ¹, associated with ether (C–O–C) and carbon–carbon (C–C) bonds in the pectin ring structure. However, other authors have found minimal spectral differences between low- and high-methoxyl pectins, suggesting that the degree of esterification may not always produce substantial shifts in this spectral region (Chaiwarit et al., 2020).

In this study, the SEM micrographs (Figure 4) revealed clear morphological differences between control and active films, reflecting the structural impact of incorporating lactocin AL705. While the HMP/Control films exhibited a smooth and compact surface, the inclusion of the bacteriocin resulted in a rougher morphology, without evidence of aggregation. Similar surface alterations associated with the incorporation of antimicrobial agents have been reported in active gelatin films containing nisin (Abarca et al., 2022). In contrast, Gebrechristos et al. (2020) observed that the addition of potato peel aqueous extract to potato starch films decreased surface homogeneity and induced crack formation, highlighting that the type and compatibility of the active compound strongly influence the final film microstructure.

Finally, to evaluate the antimicrobial effectiveness of the active films intended for food packaging, a challenge test was performed. L. monocytogenes was completely inhibited in cooked ham samples coated with HMP/AL705 films for up to seven days at both 4 and 10 °C, whereas the HMP/Control films showed no inhibitory effect against the pathogen. These results confirm that the antimicrobial activity is attributable to the presence of lactocin AL705 in the active films, as the control films remained inactive under comparable pH conditions. Our findings are consistent with those reported by Yang et al. (2020), who observed complete inhibition of L. monocytogenes for seven days at 4 °C using CNF/nisin hybrid films applied as liners in low-density polyethylene packaging for ready-to-eat ham. A similar inhibitory effect has also been reported for ham coated with poly-(lactic acid)–cellulose nanocrystal films containing nisin (Salmieri et al., 2014). However, several other studies have reported only limited anti-listerial activity when edible films incorporating antimicrobial agents are applied to refrigerated meat products (Beverly et al., 2008; Degli Esposti et al., 2018; Ravishankar et al., 2012; Xie et al., 2018). Degli Esposti et al. (2018) reported greater antimicrobial activity against Listeria monocytogenes in chicken samples stored at 22 °C than at 4 °C when using live- Enterococcus -doped films over 14 days. Edible chitosan coatings were effective in reducing Listeria monocytogenes ATCC 19117 populations in ready-to-eat roast beef stored at 4 °C, achieving reductions in Listeria growth of between 2 and 3 log CFU/g compared with control samples (Beverly et al., 2008). In comparison with our results, smaller reductions in Listeria populations have been reported in ham and mortadella samples coated with films activated with 3% carvacrol and stored at 4 °C for 7 days.

The incorporation of lactocin AL705 into HMP-based films results in materials with enhanced biological activity while maintaining acceptable mechanical, physical, and optical properties. The addition of the bacteriocin did not alter the chemical structure of the polymer but contributed to reduced film permeability, likely due to increased interactions between the HMP matrix and lactocin AL705. Notably, the incorporation of lactocin AL705 enabled the complete inhibition of Listeria monocytogenes FBUNT in cooked ham stored at 4 °C and 10 °C for 7 days. Based on its effective anti-listerial activity, combined with stable technological characteristics, this study highlights the potential of lactocin AL705 incorporated into HMP films as a novel alternative for preserving ready-to-eat refrigerated products.