1. Introduction
The United Nations General Assembly set a goal for sustainable development in 2015 to fight against hunger, which includes ensuring food security [
1,
2]. However, one of the major problems that threaten food safety is the formation of biofilms and the spread of pathogenic bacteria in the premises for the production and storage of food products, as well as on equipment for its transportation [
3,
4,
5,
6,
7]. Biocidal coatings are coatings made to stop the growth of bacteria or eliminate them [
8,
9]. The use of biocidal coatings on food contact surfaces, equipment, and packaging is an effective way to prevent the growth and spread of harmful microorganisms [
8]. These coatings contain antimicrobial agents, such as silver ions, organic antibiotics such as chlorohexidine, etc., which work by attacking the cell walls of microorganisms and preventing their growth [
8,
10,
11,
12,
13].
Salmonella, Listeria and
Escherichia coli are just a few of the major foodborne pathogens that biocidal coatings have been proven to be effective against [
14,
15]. Overall, the use of biocidal coatings in the food industry is an effective and important measure to reduce the risk of foodborne infections. It can help protect the health and well-being of consumers and also reduce the economic impact of foodborne illnesses.
Although it is normal procedure to treat production facilities with formulations based on conventional antibiotics, it may not always lead to the desired result due to the low adhesion of the biocides to the surfaces being treated and to the rapid development of bacterial resistance [
16,
17]. Therefore, there is a need for new, affordable and cheap antibacterial compositions that can be effectively used in food factories and shops. To tackle this issue, polymers have been used as new antibacterial functional coatings. In general, polymers are usually the matrix for low-molecular-weight biocides, which provide durability but do not have an antibacterial effect by themselves [
18,
19]. One specific class of polymers that have the potential to be used as biocidal coatings are the so-called biocidal polymers, which are macromolecules with functional groups that provide antibacterial action in each monomer unit [
20,
21,
22]. Among this class of polymers, polycations are of particular interest. Polymers with quaternized amino groups were reported to be effective non-specific biocides with serious benefits compared to conventional low-molecular-weight antibacterial agents. Polycations do not cause the development of induced tolerance of the bacteria and do not give rise to mutant species [
23]. Among commercially available polycations, polydiallyldimethylammonium chloride (PDADMAC) is of great potential [
24,
25]. One notable advantage of using PDADMAC as a biocide is its relatively low toxicity to humans and the environment. Quaternized polyethyleneimine (q-PEI) is the product of alkylation of a widespread polymer, polyethyleneimine (PEI), which was also admitted as effective biocide [
26]. These both polymers are completely charged in a wide range of pH that supports their high antibacterial activity independently of the pH of surrounding media. Nevertheless, the initial PEI with primary and ternary amino groups was also reported to demonstrate antimicrobial activity [
27,
28]. The mechanism of the biocidal action of the polycations is still under discussion. The antibacterial activity of polycations is primarily due to their ability to disrupt the bacterial cell membrane and cause cell death [
29]. Polycations are believed to interact with bacterial membranes through electrostatic interactions, forming a complex with the bacterial cell wall or membrane, leading to membrane destabilization. Once the bacterial membrane is disrupted, polycations can enter the bacterial cell and bind to intracellular molecules such as DNA and proteins, leading to further cell damage and eventually cell death.
PEI and PDADMAC have been shown to be effective against number of pathogens such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus subtilis. They have also been found to be effective against antibiotic-resistant strains of these bacteria.
Overall, the antibacterial activity of PDADMAC and PEI makes them promising candidates for use in a variety of applications, including food processing and packaging.
The average molecular weight and molecular weight distributions of polymers are key parameters that determine the physical and mechanical properties of the materials [
30,
31,
32]. Due to the polymerization technique used to produce them, commercial samples of PEI and PDADMAC have high polydispersity. More accurate and regulated polymerization processes should be used to control the molecular weights and their distribution [
33]. However, this could make getting polycations on an industrial scale more difficult and reduce their commercial availability.
Therefore, the key task is to establish the role of the molecular weight of polycations to determine the optimal degrees of polymerization required to create stable and effective antibacterial coatings. In the first part of this paper, we focus on exploring the main properties of coatings based on PDADMAC and PEI with different molecular weights and make a recommendation on the choice of the degree of polymerization for creating stable coatings. The second part is dedicated to the study of the biocide’s efficiency against foodborne Gram-positive bacteria of the coatings from the optimal samples of the polycations.
4. Discussion
Polymer coatings are widely used as protective layers to prevent formation of biofilms on different surfaces [
38,
39,
40]. In general, these protective polymers could be divided in classes of anti-fouling coatings, bacteria-killing coatings and matrixes for distribution of low-molecular-weight biocides. The first ones prevent the adsorption of the bacteria on the surface of the treated material, while the second interact with microorganisms, causing disorders in their functionalization. Polycations commonly act as biocidal molecules. One of the proposed mechanisms of action of polycations is their interaction with the negatively charged membrane of the bacterial cell due to their own positive charge [
40,
41]. As a result, this leads to structural rearrangements in the lipid bilayer and disruption of the vital activity of bacteria or their deaths [
42]. L.D. Melo et al. report that PDADMAC tends to cause membrane stress in bacterial cells, which can ultimately lead to the destruction of cellular structures [
43]. PEI is known to contain a sufficient fraction of protonated amines that can bind to negatively charged bacterial cell surface components, which can lead to cell depolarization, cell wall/membrane disruption, and cell lysis [
44]. It was found by lkka M. Helander that when PEI interacts with a culture of gram-negative bacteria in solution, it permeates their outer membrane, making the bacteria more susceptible to the environment, including additionally added antibiotics [
45]. There is also evidence that polycations can affect the stage of protein biosynthesis (translation) [
46]. However, the complete mechanism of the antibacterial action of polycations is still under investigation. The presence of a positive charge on the coating cannot ensure a biocidal effect, as was demonstrated by Sorzabal-Bellido and colleagues: polydimethylsiloxane with a surface layer modified with primary amino groups did not cause a significant antibacterial effect [
47]. It seems that not only a charged layer but a number of cationic units on a flexible chain should exist on a biocidal coating. Enhancement of the antibacterial action of polyelectrolyte coatings could be achieved with the use of additional biocides of non-polymer nature and anti-fouling macromolecules. When it comes to biocides, the question of the impact of such substances on humans is important. PDADMAC is one of the brightest representatives of biocidal polycations which are allowed for direct contact with humans. It has also found its medical uses, as a biocidal additive in dental materials, and as a component of wound dressings [
48,
49]. At the same time, PEI is approved by the FDA for use as an indirect food additive [
50].
Both polycations, PDADMAC and PEI, were shown to possess antimicrobial activity towards
B. subtilis in solutions. Two important observations should be pointed out. The nature of an amino group in a polycation and the degree of polymerization do not play essential roles in the antibacterial activity of the studied polymers in solutions. Taking into account previously reported data on MIC of a hyperbranched copolymer of epichlorohydrin and ethylenediamine towards
B. subtilis with values of a similar order, we may state that a linear or hyperbranched structure of the macromolecule cannot be considered as a key parameter affecting the biocidal activity of polycations [
46].
The application of an aqueous solution of polycations to the surface of a hydrophilic glass with further drying in the air leads to the formation of a polymer coating. The adhesion of the macromolecules on the glass is driven by electrostatic forces between negatively charged silanol groups and amino groups. Most of the macromolecules in an adsorbed layer form a film of interpenetrated chains. Polycations are known to be hygroscopic. Therefore, it is reasonable to expect that polycation films will absorb water from the environment. In solutions, solvation of the ammonia salts depends on the nature of the amino groups, but for the studied films of polyelectrolytes no significant influence of the primary, ternary and quaternary amino groups on swelling in an environment with controlled humidity was found.
It is obvious that degree of polymerization of macromolecule should affect the strength of adhesion on a glass surface. The dynamometric experiments have shown that the mechanical break of the polyelectrolyte films is predominantly governed by cohesion forces. The shape of the stress curves in
Figure 5 reflects some correspondence with a cohesion break mechanism. For the PEI samples with high molecular weights, the peak stresses were almost identical, with a mean value of 18,500 Pa, and a decrease in the mechanical properties of films was observed for an oligomer fraction with a mean value of peak stress of 11,600 Pa. The same behavior of the mechanical properties was observed in the films from different samples of PDADMACs. For the samples with high molecular weights, the peak stresses were almost identical, with a mean value of 30,800 Pa, and the decrease in the mechanical properties of films was observed for an oligomer fraction with a mean value of peak stress of 26,600 Pa. These results are in good agreement with the cohesion-governed mechanism of the film break. Moreover, the differences in absolute values of peak stresses between PEI and PDADMAC macromolecules could be attributed to differences in macromolecule architectures. Linear PDADMAC macromolecules could penetrate between many lateral layers inside the film, while for the branched PEI molecules this possibility is restricted.
The vanishing of the polyelectrolyte coating with water depends on two major parameters. First, the nature of the amino group: for the quaternary amino groups in PDADMAC, the process of the dissolving of the film takes place faster than for the PEI with primary and ternary groups. Second, the molecular weight of macromolecules: with the reaching of critical values of molecular weights of polycations, the films undergo fast mass loss under the wash-off procedure. For the PDADMAC molecules, this critical weight was 100 kDa, while for the PEI this value was 40 kDa. Both polycations have different masses of monomer units and different architectures. Therefore, such parameters as “degree of polymerization” and “average molecular weight” could not be used for direct comparison of the results for these polycations. Nevertheless, we have demonstrated that the diffusion coefficient of the polycation is the parameter that allows us to describe correctly the behavior of the films in the wash-off procedure. The critical value of the D0 = 1 × 10−7 cm2/s determines the resistance of the polyelectrolyte film towards fast wash-off for both PEI and PDADMAC.
Despite it being reported that an increase in the molecular weight of the polymers of the same structure could decrease or increase their biocidal activity [
51,
52,
53], we have demonstrated that the molecular weight of the polycation has a greater effect on the mechanical properties of the films and their behavior under watering conditions. Only oligomers of PEI with a mass of 2 kDa and less demonstrated a decrease in biocidal activity. Therefore, polycations with high molecular weights have a higher potential for the preparation of effective biocidal coatings.
Thus, the efficiency of polycationic coatings towards foodborne bacteria L. monocytogenes was analyzed using the samples with higher values of Mw: PEI-750 and PDADMAC-500. The morphology of the coatings obtained by AFM were confirmed to be above the coating. Hence, bacteria with negatively charged membranes will adsorb on the film and undergo the action of the polycations. At the same time, the macromolecules that leave the surface of the film during the watering process could act as biocides in suspension of the bacteria that were not adsorbed on the film. Both PEI and PDADMAC were demonstrated to have antibacterial activity in solution and on the surface of films against L. monocytogenes. Therefore, the choice of the polycation and its molecular weight for the formation of the biocidal coatings should be determined more by requirements than by the mechanical properties of the supposed coating.
5. Conclusions
Branched PEI and linear PDADMAC of different molecular weights were studied to estimate the roles of the chemical nature of the charged groups, the architecture and the degree of polymerization of macromolecules in the physical–mechanical and antibacterial properties of the coatings from these polymers on glass surfaces. Surprisingly, the values of MIC for polyelectrolytes did not depend on molecular weights of the polymer samples in a wide range of the masses. The chemical nature of the amino group (primary, ternary or quaternary) was not found to possess a significant impact on the biocidal properties of macromolecules. For the coatings from the different samples of PEI and PDADMAC, it was demonstrated that saturation of the films with water depends on the humidity of the surrounding media but not on the structural and chemical characteristics of the polycations. The mechanical properties of the coatings from polycations were demonstrated to have a determining cohesive nature between macromolecules over adhesion forces between surface and polymer film. Linear PDADMAC ensures a higher cohesive strength due to the possibility of penetrating between more layers in films than branched PEI. With the decrease in molecular weight, the mechanical stress required to break the coating is reduced for both series of PEI and PDADMAC. The ability of the polyelectrolyte film to resist wash-off with water strongly depends on the diffusion coefficient of the macromolecules that form the coatings. This parameter is more suitable for describing the decisive characteristics of macromolecules that allows one to compare polyelectrolytes with different architectures and chemical structures. Thus, the polycations with high molecular weights have higher potential for utilization in formation of coatings. Concerning the biocidal activity of the polycationic films, the antibacterial effect towards L. monocytogenes was demonstrated for both PEI and PDADMAC with no significant difference.
Therefore, the choice of the polycation for the effective biocidal coatings should be determined by the requirements for the mechanical properties of the films. These properties are affected by the diffusion coefficients, architectures and chemistry of macromolecules. At the same time, the antimicrobial activity of the coatings is determined by the polycationic nature of the coatings without a significant role being played by the nature of the amino group.