Development and Assessment of a Nisin/Chitosan Composite as an Antibacterial Coating for Polyester Fabric

Introduction

In recent years, the importance of antibacterial coatings has gained growing recognition across multiple industries, including healthcare, food packaging, and textiles. The need for effective measures to prevent the proliferation and transmission of harmful bacteria has become paramount to ensure product safety and minimize the risk of infectious diseases. As a result, the development of antibacterial coatings has become essential for maintaining hygienic conditions and enhancing the overall quality of various products (1, 2). Among the vast array of antibacterial agents currently available, nisin and chitosan have emerged as promising candidates due to their natural origins, broad-spectrum antimicrobial activity, and biocompatibility (2). Polyester fabric is extensively employed in various medical and food-related applications due to its desirable properties, such as lightweight, durability, and ease of processing (3). However, a significant drawback of polyester fabric is its susceptibility to bacterial colonization, which can lead to contamination and compromise its hygienic use. This vulnerability underscores the urgent need to develop effective antibacterial coatings specifically tailored for polyester fabrics (4).

The traditional antibacterial methods using chemical disinfectants and metal-containing coatings also present common shortcomings, such as, toxicity, ecosystem degradation, and constrained durability (5). Hence, the development of innovative, non-toxic, and eco-friendly antibacterial coating materials for polyester textiles is imperative.

Although existing literature has discussed various antibacterial coatings, such as silver nanoparticles (5, 6), quaternary ammonium compounds (7), and antimicrobial agents of natural origin (8), the literature on biopolymer-based composite coatings for polyester textiles is lacking.  The literature on biopolymer-based composite coatings for polyester textiles is lacking. Additionally, the application of nisin, a naturally derived antimicrobial peptide, combined with chitosan as a textile coating, is not well-researched. Hence, an identified gap in the literature lends itself to supporting the novel combination of nisin and chitosan for the production of the composite coating optimally designed for application onto polyester textiles, being potentially non-toxic and ecologically sustainable.

The use of nisin, renowned for its potent antimicrobial activity against a wide range of Gram-positive bacteria, offers an opportunity to combat the bacterial colonization commonly observed on polyester fabric. The mechanism of action of nisin involves disrupting the integrity of bacterial cell walls, leading to cell death (9, 10). This ability to selectively target and eliminate harmful bacteria makes nisin an ideal candidate for incorporation into antibacterial coatings.

Chitosan has unique attributes as a composite coating. Derived from the deacetylation of chitin, chitosan (Cs) exhibits exceptional antimicrobial activity against Gram-positive and Gram-negative bacteria (2). Its mode of action involves interfering with the structure and function of bacterial cell membranes, resulting in the inhibition of bacterial growth. Furthermore, the biocompatibility and biodegradability of chitosan make it a suitable component for contact with biological systems, ensuring its safety and environmental compatibility (8, 11).  Combining nisin and chitosan in a composite coating provides synergistic antimicrobial effects, improved stability, and controlled release of antibacterial agents (12). This composite coating aims to create a protective barrier on the surface of polyester fabrics and actively inhibits bacterial colonization and reduces the risk of contamination.

The primary objectives were to determine the structural properties of N/Cs coatings using SEM and FTIR, and to investigate their antimicrobial ability against Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Listeria monocytogenes. The stability of the coating after washing was then determined. Cytotoxicity testing was furthermore carried out to aid biocompatibility studies.

 Materials and Methods

Bacterial strains, media, and materials

In this investigation, the following bacterial strains were utilized: Escherichia coli PTCC1395, Pseudomonas aeruginosa PTCC 1074 (Gram-negative), Staphylococcus aureus PTCC1431, Bacillus cereus PTCC1015, Listeria monocytogenes ATCC19112, and Enterococcus faecalis PTCC 1237 (Gram-positive). These strains were obtained from the culture collection of the I.R. Dept. (Tehran, Iran). Nisin was purchased from Sigma. The fabric used in this study was made of 100% polyester textile. It had a plain-woven structure with a filament yarn of 150 denier and a weight of 140 grams per square meter. The fabric was produced by Yazd Baf Yazd Company. Chitosan was synthesized from shrimp shells in a previous study (12).

 Method

Finishing treatments were performed under controlled laboratory conditions. Polyester fabrics were immersed for 3 min in a solution containing 2.56 mg/mL nisin and 1.5% (w/v) chitosan, prepared in 1% (v/v) acetic acid. Subsequently, the treated fabrics were processed using a laboratory foulard machine (Model DM-450, Tsujii Dyeing Machines MF6 Co., Ltd., Japan) at ambient temperature, ensuring a 100% wet pick-up—defined as the uptake of solution equivalent to the fabric's own weight. The samples were then dried at 100 °C and thermally cured in an oven at 150 °C for 2 min (13, 14).

 Characterization of the Coated Fabrics

The surface structure of the coated fabrics was analyzed using a scanning electron microscope (SEM) (Phenom ProX Desktop SEM - Thermo Fisher Scientific, USA). Furthermore, the presence of nisin and chitosan (N/Cs) in the coated fabrics was confirmed using Fourier transform infrared (FTIR) spectroscopy (Shimadzu IRPrestige-21 spectrometer) (13, 15).

The antimicrobial properties of uncoated and coated polyester textile samples were evaluated using the agar diffusion method. Overnight cultures of E. coli, P. aeruginosa, S. aureus, B. cereus, L. monocytogenes, and E.  faecalis (10⁷ cells/mL) were individually inoculated onto Tryptic Soy Agar (TSA) plates. Fabric samples were placed on the inoculated plates and incubated at 37 °C for 24 h. The inhibition zones surrounding the samples were measured using a ruler. All experiments were conducted in triplicate, and the mean values were recorded (12, 14, 16). 

The durability of the antibacterial activity was evaluated after subjecting the samples to 10 washing cycles at 60 °C, at 15 rpm, for 15 min, using a nonionic surfactant called Golvash (Siavashn Company, Yazd, Iran) at a liquor-to-fabric ratio of 30:1.

To evaluate the antimicrobial activity of the sample fabrics (uncoated and coated polyester textiles), the following liquid culture test method was employed. First, the fabric samples were sterilized using ultraviolet (UV) light for 1 h. The antimicrobial activity was assessed using a standard shaking flask (ASTM E2149-01) recommended for fabrics with immobilized permanent active agents. Polyester fabrics were individually incubated with 5 ml of E. coli, P. aeruginosa, S. aureus, B. cereus, L. monocytogenes, or E. faecalis bacterial suspension at 37 °C at 230 rpm, respectively. The initial bacterial concentration in each test was approximately 10⁷ cells/ml.

To determine the antimicrobial properties, samples were collected from the test tubes before and after 24 h of contact with the fabric samples. The liquid cultures were serially diluted in a sterile buffer suspension, plated onto tryptic soy agar (TSA), and incubated at 37 °C for 24 h to determine the number of surviving bacteria. The antimicrobial activity, expressed as the percentage of bacterial reduction, was calculated by comparing the number of viable bacteria before and after contact with the coated textiles using the following formula:

Bacterial reduction (%) = ((A − B)/A) × 100

The mean number of bacteria before contact with the coated textiles is (A), while the mean number of bacteria after contact is (B) (15, 17).

 Evaluation of Washing and Fixation Effects on Antimicrobial Performance

The durability of the antibacterial activity was measured by subjecting the samples to 10 washing cycles at 60 °C and 15 rpm for 15 min using a 2% nonionic surfactant Golvash (Siavashn Company, Yazd, Iran) (15).

To test the durability of the hybrid N/Cs coating under laundering conditions, antimicrobial inhibition rates were measured before and after washing for each bacterial strain. The absolute change in inhibition percentage values (pre-wash vs. post-wash) reflects the change in performance, whereas the retention percentage, calculated as (post-wash inhibition / pre-wash inhibition) × 100, provides a measure of the stability of antimicrobial performance after washing. Retention values below 100% indicate degradation of the coating and a proportional decrease in performance, whereas values above 100% could indicate an improvement in post-wash activity, potentially due to experimental variability or molecular reconfiguration of the coating. These measures provide valuable descriptors of the robustness of the coatings and an understanding of their suitability for applications involving repeated-use textiles.

In the same context, the effect of fixation on antimicrobial performance was estimated by comparing the inhibition rates of standard N/Cs-coated materials and those subjected to fixation. The absolute difference (standard-N/Cs coated and fixed- N/Cs coated materials) accounts for the magnitude of the alteration in efficacy performance, whereas retention percentages [((fixed N/Cs coated inhibition / standard N/Cs coated inhibition) × 100] serve as antimicrobial stability or improvement measures after fixation. Values higher than 100% indicated an improvement in activity performance, whereas values below 100% indicated a decrease in performance.

 Cytotoxicity evaluation

The development and proliferation of cells on various fabric samples (uncoated and coated textiles) were evaluated using the colorimetric MTT method after 7 days. Initially, the coated and uncoated fabric samples were placed in 24-well plates and sterilized with UV light for 1 h. Following the plates were then incubated with 3 mL of complete growth medium (DMEM) containing human foreskin fibroblasts (Fhh: Passage 18) sourced from the Biotechnology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadougi University of Medical Sciences, Yazd, Iran. The mixture was then incubated in a CO₂ incubator at 37 °C for either 1 or 7 days. Ultimately, the colorimetric MTT method was employed to assess the development and proliferation of fibroblasts on the different coated and uncoated fabric samples. At designated time intervals, a solution containing 5 mg/ml of mitochondrial reagent in phenol red-free RPMI 1640 (10% v/v) was added to each well and incubated in the dark for 4 h. After incubation, the culture medium was removed by aspiration, and the dye was stabilized overnight using acidic isopropanol (0.04–0.1 N HCl in absolute isopropanol). The results were quantified using an ELISA microplate reader (Fluostar Optima, BMG Lab Technologies, Germany) at a wavelength of 570 nm, with background signals subtracted at 630 nm.

 Statistical analysis

Statistical analyses were performed using SPSS software (version 19.0). All experimental procedures were conducted in triplicate, and the resulting data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was employed to evaluate statistical differences among groups, followed by Duncan’s multiple range test to determine significant differences between mean values. A significance threshold of p < 0.05 was applied throughout the analyses.

 Results

Characterization of the coated fabrics

SEM images of the treated and untreated polyester fabrics clearly reveal that the coating is evenly spread over the fabric surface, indicating uniform formation (Fig. 1).

Fig. 1. Scanning electron microscopy (SEM) photomicrographs of (a) uncoated polyester fabric, (b) polyester fabric coated with chitosan (Cs), and (c) polyester fabric coated with nisin/chitosan (N/Cs).

FTIR analysis was performed to determine the presence of N/Cs on polyester fabric. The spectra obtained from the different antimicrobial coatings exhibited similar patterns (Fig. 2). In the Cs-coated samples, broad peaks were observed at 3400 cm^-1 and 1030–1155 cm^-1, corresponding to the stretching of O-H and N-H bonds, respectively, as well as C-O bonds. Furthermore, absorption peaks were detected at 2850–2950 cm^-1, approximately 1550–1650 cm^-1, and 1400 cm^-1, corresponding to C-H stretching, amine groups, and carboxyl groups, respectively. Polysaccharide bands (C-O-C stretching) at 1060 cm^-1 were also identified (18). 

Fig. 2 illustrates the FTIR spectra of (a) uncoated polyester fabric, (b) polyester fabric coated with chitosan (Cs), and (c) polyester fabric coated with nisin/chitosan (N/Cs).

Table 1. displays the inhibition zone diameters of the coated polyester fabric, fixed-coated polyester fabric, and washed-coated polyester fabric in the presence of E. coli, P. aeruginosa, S. aureus, B. cereus, L. monocytogenes, and E. faecalis.

The addition of nisin to the Cs-coated fabric resulted in noticeable changes in the FTIR spectra. A new absorption peak appeared at 3,150–3,450 cm^-1, indicating the presence of the -OH group in the peptides. Additional peaks were observed at 1,656 cm^-1 and 1,537 cm^-1, which are predominantly associated with the amide I and amide II bands, respectively. These spectral modifications can be directly attributed to peptide linkages within the fabric (18, 19). 

 Antimicrobial Performance and Fixation Effects

The antibacterial effectiveness of different polyester fabric samples (coated polyester fabric, fixed-coated polyester fabric, and washed-coated polyester fabric) was evaluated against various bacteria, including E. coli, P. aeruginosa, S. aureus, B. cereus, L. monocytogenes, and E. faecalis, compared to that of polyester fabric (Table 1, Fig. 3). The results indicated that the coated, fixed-coated, and washed-coated polyester fabrics with N/Cs did not exhibit any inhibitory effects on L. monocytogenes or E. faecalis (p ≥ 0.05). However, these fabrics exhibited inhibitory effects on E. coli, P. aeruginosa, S. aureus, and B. cereus bacteria (p ≤ 0.05).

 Fig. 3 shows the antibacterial activity of the coated, fixed-coated, and washed-coated polyester fabrics against (a) B. cereus, (b) S. aureus, (c) E. faecalis, (d) L. monocytogenes, (e) E. coli, and (f) P. aeruginosa after 24 h of contact.  (p-value < 0.05). Distinct English letters above each graph indicate statistically significant differences, as determined by Duncan's multiple range test at the 5% significance level.

Next, we assessed the antibacterial efficacy of the N/Cs hybrid polyester fabric against six medically significant bacterial species in broth media (Fig. 3). The N/Cs coating of polyester fabrics exhibited strain-specific antimicrobial activity, with inhibition percentages significantly decreasing bacterial growth compared with controls without coating.  B. cereus exhibited complete inhibition of growth (100%) after treatment with N/Cs, an improvement of 44.45% over the control (55.55%). E. coli exhibited significant improvement with 47.07% inhibition, an improvement of 24.85% over that of the control (22.22%). S. aureus and E. faecalis exhibited poor improvements of 18.78% and 20.09%, respectively, while L. monocytogenes exhibited only a slight gain of 6.12%. Comparatively, however, P. aeruginosa exhibited a very poor coating effect with only a 2.75% improvement in inhibition (53% vs. 50.25%). These results demonstrate the synergistic antimicrobial activity of nisin and chitosan, with optimal activity against B. cereus (p ≤ 0.05).

Fixation of the N/Cs coating showed heterogeneous outcomes between bacterial strains, with retention percentages ranging from 68.30% (E. faecalis) to 122.09% (P. aeruginosa), with an average of 93.6%. There was increased post-fixation activity with P. aeruginosa and L. monocytogenes (p ≤ 0.05), while diminished efficacy was observed among other Gram-positive species (Table 2 and Fig. 3).

Table 2. Comparison of antimicrobial efficacy of unfixed and fixed nisin-chitosan (N/Cs) -coated polyester fabrics.

Table 3. Comparison of antimicrobial efficacy of unwashed and washed nisin-chitosan (N/Cs) coated polyester fabrics.

 Effect of Washing on Antimicrobial Performance

The effect of washing on the antimicrobial activity of the hybrid N/Cs coating was evaluated by determining the percentage of bacterial inhibition before and after washing against some strains. Washing tended to cause a loss of antimicrobial activity, with retention values of 65.16% for E. faecalis and 106.85% for P. aeruginosa, with an overall retention of approximately 79.8%. The highest material loss was seen in Gram-positive bacteria, more so in B. cereus and E. faecalis, and the least in variable responses by Gram-negative strains (Table 3, Fig. 3) (p ≤ 0.05).

 Cytotoxicity Assessment

To determine the biocompatibility of the N/Cs-coated textiles for medical applications with direct skin contact, cytotoxicity was examined via the MTT assay on fibroblast cultures (Fig. 4). The optical density (OD) of the uncoated polyester material (0.850) corresponded with the cell control (0.900), indicating low cytotoxicity and only a small loss in cell viability of 5.6%. The N/Cs-coated samples recorded an OD of 0.715, which was close to that of only Cs-coated fabrics, and a 20.6% reduction in cell viability. Statistical analysis did not reveal a significant difference between the coated and uncoated samples (p ≤ 0.05), reaffirming that the hybrid N/Cs coating maintains acceptable cytocompatibility for possible future use in biomedical textiles.

Fig. 4. illustrates the cytotoxic activity of the coated, fixed-coated, and washed-coated polyester fabrics towards fibroblast cells after a 7-day contact period.   (p-value < 0.05). Distinct English letters above each graph indicate statistically significant differences, as determined by Duncan's multiple range test at the 5% significance level.

Discussion

The textile industry has witnessed substantial growth in the development of medical, healthcare, and hygienic products, driven by the need for products that can meet high standards of performance, such as mechanical strength, elasticity, breathability, and moisture transfer. Natural fibers are biocompatible, but they often lack the endurance required for long periods of clinical use; thus, synthetic fibers such as polyester (20) are increasingly preferred.

Polyester, a commonly employed synthetic polymer characterized by recurring ester units obtained from terephthalic acid and ethylene glycol, is valued for its considerable tensile strength, resistance to chemicals, and economical manufacturing process (21, 22).  However, its hydrophobic nature creates challenges for the functionality of the surfaces, primarily during antimicrobial finish treatments that medical textiles require (23).

This study demonstrates the effective preparation of hybrid N/Cs coatings on polyester textiles to provide antimicrobial functionality with potential use in clinics and hygiene. SEM characterization confirmed even coating coverage, an important requirement achieving reliable antimicrobial activity and restricting bacterial colonization (Fig. 1). FTIR spectroscopy showed characteristic peaks of hydroxyl, amino, and amide groups, with new bands at 3150–3450 cm^-¹, 1656 cm^-¹, and 1537 cm^-¹ by the incorporation of nisin, suggesting the formation of peptide linkages and integration synergies (Fig. 2) (18, 19).

Dilution tests of the broth revealed that the N/Cs-coated textiles acted differently against various strains (p ≤ 0.05; Fig. 3). The absolute inhibition of B. cereus (100%) was 44.45% higher than that of the control, owing to the synergistic effect of chitosan and nisin activity. There were impartial improvements in E. coli (+24.85%), E.  faecalis (+20.09%), and S.  aureus (+18.78%). There were slight improvements for P. aeruginosa (+2.75%) and L. monocytogenes (+6.12%), demonstrating how Gram-negative bacteria resist treatment (24, 25) (Fig. 3).

Diffusion tests on agar revealed no zones of inhibition against L. monocytogenes and E. faecalis. This is probably because active compounds do not propagate effectively on solid media. However, broth tests revealed a considerable decrease in viability due to direct exposure (26). 

The fixation of the N/Cs coating yielded heterogeneous outcomes across bacterial strains, with retention rates ranging from 68.30% for E. faecalis to 122.09% for P. aeruginosa, with an overall mean of 93.6%. Significantly elevated post-fixation activity was observed for P. aeruginosa and L. monocytogenes (p ≤ 0.05), whereas reduced efficacy was noted against other Gram-positive species (Table 2 and Fig. 3). These findings indicate that fixation can enhance antimicrobial activity to a certain extent; however, careful optimization of the fixation conditions is essential to minimize activity loss and ensure consistent efficacy across a broad range of microbial targets (27). 

Despite advancements in antimicrobial fabric development, hospital textiles remain susceptible to contamination during their use. Hence, frequent washing is necessary to avoid the proliferation of pathogens (28).

Washing endurance tests showed an average retention of 79.8%, with considerable loss of B. cereus (67.74%) and E. faecalis (65.16%), probably because of leaching of water-soluble nisin and partial loss of chitosan through erosion of the coating. Conversely, P. aeruginosa experienced a slight gain (106.85%), possibly owing to the reconfiguration of the coating following washing. These results indicate that new encapsulation or covalent bonding strategies should be developed to enhance washing resistance (27, 29).

Various natural antimicrobial agents, such as plant extracts, natural dyes, honey, and essential oils, have been evaluated for fabric functionalization (28, 30). 

Amphiphilic cyclodextrins offer environmentally friendly finishing solutions (31), whereas lignin-based coatings derived from sugarcane bagasse are effective against Staphylococcus epidermidis (32).

Chitosan is a significant biopolymer with antimicrobial and odor-repelling properties. It can eliminate bacteria such as S. aureus, E. coli, and L. monocytogenes through its hydrogel (33).  Nevertheless, its success will rely on the substrate and formula being applied, particularly, as studies conducted on silica sol–chitosan composites revealed (16). Furthermore, pectin- and chitosan-based AgNP films exhibited significant antibacterial activity and can be used for food packaging (6).

Mirhosseini et al. (2023) recorded the persistent antimicrobial activity of chitosan–nisin coated on cotton fabrics, remaining even after facing ten wash cycles (34). This durability is attributed to the hydrophilic and cohesive structural properties inherent to cotton substrates (35).

Different physical strategies have been utilized to produce antimicrobial polyester fabrics, including plasma treatment, which is one of them. Plasma treatments involve the use of surface coatings that are cost-effective and environmentally friendly. Alloys ensure reliable coverage and compatibility with many types of materials and retain the inherent characteristics of the substrate (36).

Cytotoxicity tests employing human foreskin fibroblast cells—a dermal safety model—found negligible effect, with only a decrease of 20.6% in the viability of cells of N/Cs-coated textiles (p ≥ 0.05; Fig. 4), equivalent to controls without coating (37). The GRAS (Generally Recognized as Safe) status of nisin and chitosan, and their low mammalian toxicity (18, 38), their antioxidant activity justify their safe incorporation into biomedical textiles (15).

 Conclusion

This study effectively demonstrated the formation of a consistent and functionally active N/Cs antimicrobial coating on polyester textiles. SEM and FTIR characterizations confirmed the consistent coverage of the surfaces and chemical incorporation of bioactive entities, with spectral confirmation of peptide and polysaccharide bonding. Antibacterial tests showed that the N/Cs-coated textiles have shown substantial inhibitory activity against major Gram-positive and negative pathogens, notably B. cereus and E. coli, with limited activity against L. monocytogenes and E. faecalis. Adhesion and washing evaluations showed strain-dependent antimicrobial persistence with average retention of 93.6% and 79.8%, respectively, and implied partial coating resilience under operational conditions. Notably, cytotoxicity evaluations confirmed that the N/Cs-treated textile fabrics were biocompatible and thus sustainable for medical and hygienic textile usage applications. The results have shown the potential of N/Cs hybrid coating technology to act effectively and be compatibly applied as a skin-experience antimicrobial agent, and identify necessary future improvements to achieve higher long-term stability and wider antibacterial coverage.

Acknowledgments

 Authors gratefully acknowledge the generous cooperation of the Nano-Structured Coatings Institute, Yazd Payame Noor University, PO Code 89431-74559, Yazd, Iran. We gratefully thank the people of the Biotechnology Research Center, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran, especially Dr. Habib Nikukar and Dr. Behrouz Aflatoonian, for their collaboration.

Author contributions: M.M. designed and performed experiments, analyzed data, and wrote the paper. M.A. performed experiments and analyzed data, H.M.A. designed experiments, S. S Designed experiments, and helped with writing and editing the article.