Nanoformulations for lysozyme-based additives in animal feed: An alternative to fight antibiotic resistance spread

1 Introduction

2 Use of enzymes as antimicrobial agents

Bacterial resistance to antibiotics has grown faster than the development and/or discovery of new antibiotics and thus has boosted research in this area for facing the problem. The initial efforts to find antibiotics in culturable microorganisms rapidly reached a limit after 40 years. The following endeavor, aiming to enhance known antibiotics based on either computational predictions or just combinatorial, random exploration of the so-called “chemical space” of the molecules, has also reached an impasse [38].

The development of modern high-throughput molecular tools has led to the generation of vast amounts of information, such as in genomic and metagenomic sequencing, which allows massive in silico screening. This brute-force tool could provide potential new antibiotics and also prediction of new targets, to narrow down the prospects to a small number of candidates, which can be experimentally tested. The massive data could also be fed to powerful methods such as deep learning, to come up with novel antibiotic candidates [39].

One of the drawbacks of small-molecule based antibiotics is the extraordinary ability of bacteria to self-modify through mutations, to adapt to the presence of antibiotics and overcome its effects. One area of research that has gained attention due to a lower probability of generating AR is the exploitation of antibiotic biomacromolecules, particularly wild-type enzymes. In this case, hydrolytic enzymes with an antimicrobial effect may be specifically directed to degrade various targets such as (a) proteins and polysaccharides that are components of the biofilms and/or the bacterial cell walls of pathogens [40] and (b) small molecules that allow the pathogen to thrive, such as quorum-sensing molecules. On the other hand, redox enzymes may be less specific in terms of a particular target in the pathogen, causing general damage to the pathogen cell or the biofilm created by bacteria. The biofilm formation is essential for bacterial survival and the exertion of their pathogenicity [41]. Examples of these enzymes are shown in Table 2, including native enzymes and engineered enzymes into adequate vectors (i.e., bacteriophages as in the study of Lu et al. [44]).

The usual bacterial mechanisms to generate resistance, mentioned in the previous section, are less likely to work when the antibiotic agent is an enzyme, mainly because there may be multiple targets, such as in the case of redox enzymes, and also because some of the targets, such as polysaccharides, are not as genetically malleable as proteins (Table 3). In both cases, a larger number of mutations must arise for the bacteria to become resistant, compared to small molecule-based antibiotics.

One of this antibiotic enzyme that has attracted attention for its use in several areas, particularly in the livestock and poultry industry, is lysozyme [57]. This enzyme has had a wide industrial use as a food preservative for many decades [58]; it is nontoxic and environmentally friendly due to its biodegradable nature. On the other hand, additives in animal feed have been utilized for many decades. From pre- and probiotics, to organic acids and other micronutrients as well as digestive enzymes, the design of animal feed has allowed an increased efficiency and a reduced environmental impact [59]. The use of antibiotics has been banned in several countries, despite its clear benefits as growth promoters in young animals (for example pigs and chickens), due to the risk of favoring AR and also due to consumer pressure. However, it has been observed that some antimicrobial enzymes may benefit the performance of animal production, thus paving the way for an alternative. In the next section, we will discuss examples of the beneficial use of lysozyme in animal feed and its probable mechanisms of action.

3 Use of lysozyme in animal feed as an alternative to small molecule-based antibiotics

Lysozyme (E.C.3.2.1.17) also known as muramidases or 1,4-β-N-acetylmuramidase is a monomeric, low molecular weight enzyme that has a pivotal role in the prevention of bacterial infections in animals and is widely distributed in a variety of tissues, including the liver, articular cartilage, plasma, saliva, tears, milk, and chicken egg white (Figure 1). Lysozyme hydrolyzes a glycosidic bond present in peptidoglycan, a component of bacterial cell walls, which is composed of repeating units of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG), cross-linked by peptide bridges. Specifically, lysozyme hydrolyzes the bond between carbon 1 of NAM and carbon 4 of NAG (Figure 2). Upon hydrolysis of peptidoglycan, bacterial permeability increases, leading the bacteria to burst [60]. Most Gram-positive bacteria are extremely susceptible to lysozyme because their cell walls consist of 90% peptidoglycan, whereas Gram-negative bacteria are less susceptible because of the smaller amount of peptidoglycan in their cell wall and also the presence of a protective lipopolysaccharides (LPS) layer on the outer membrane that prevents lysozyme from reaching its target [57,61]. Although the canonic antibacterial activity of lysozyme derives from the catalytic hydrolysis of the peptidoglycan, it has been demonstrated that there is another bactericidal mechanism not related to the enzymatic activity, but rather resulting from the interaction of the protein itself with certain components of the cell wall, leading to events such as membrane depolarization and permeabilization, as well as agglutination of bacterial cells, which decreases its colony-forming ability and eventually, culminates in death of the bacteria. Inactive mutants and even denatured lysozyme display bactericidal properties, which probably arise from both the cationic and hydrophobic nature of the peptide sequence, allowing its interaction with negative charges and hydrophobic molecules in the bacterial cell wall. This non-catalytic bactericidal mechanism also explains the action of lysozyme toward Gram-negative bacteria, which may not be sufficiently protected by the presence of LPS, but rather susceptible to lysozyme precisely due to the hydrophobic nature of the lipidic layer (for recent reviews on the subject, see previous studies [57,62]). The main current application of lysozyme is in the food industry, to prevent food spoilage [63]. Due to its antimicrobial properties and low toxicity, lysozyme has attracted considerable attention to be potentially utilized in many other areas (for an updated review refer the study by Ferraboschi et al. [57]) including the animal feed industry [64,65].

Figure 1

Lysozyme from Gallus gallus (PDB 1LYZ). Catalytic residues (Glu 35 and Asp 52) are highlighted.

Figure 2

Schematic representation of peptidoglycan, present in the bacteria cell wall and the target of lysozyme. The monomer (NAM–NAG disaccharide, covalently linked to a short peptide) forms linear chains that crosslink through the peptide moieties. The site of lysozyme cleavage, the β(1-4) glycosidic bond between NAM and NAG, is highlighted.

In the livestock and poultry industry, sustained animal production is affected by various factors, and optimal gastrointestinal functionality is one of the most important. The effective functioning of the gastrointestinal tract (GIT) plays a key role in determining animal performance such as growth, milk production, as well as meat and egg quality. Optimal gastrointestinal functionality (gut health) is defined as a steady state where the microbiome and the intestinal tract exist in symbiotic equilibrium, allowing the maintenance of key physiological functions resulting in improved health [66]. The composition and metabolic activity of the GIT microbiota, which evolves throughout the life of the animal, has a significant impact on the health of the host due to several positive functions, including energy recovery from the metabolism of non-digestible components of foods, protection from pathogens, and modulation of the immune system [67,68]. Evidence that a lysozyme-supplemented diet is beneficial for the GIT microbiota has been reported in several works on mammals such as piglets, broilers, rabbits, and ruminants, and also in different species of fish (Table 4).

Xu et al. [78] evaluated the effect of different dosages of lysozyme (30, 60, 90, and 120 mg lysozyme/kg) on growth performance in weanling piglets. Their results showed that piglets fed a diet including 90 mg/kg lysozyme had greater (p < 0.05) average daily gain (ADG) (418.57 g/day) which was 12.6% higher (p = 0.105) than the control group (365.11 g/day). Also, supplementing 90 mg lysozyme/kg was as effective as antibiotics for improving the growth performance of weanling piglets: a daily dosage of 20 mg colistin sulfate plus 50 mg kitasamycin per kg resulted in ADG = 414.43 g/day. In another evaluation, the liquid diet of 10-day old piglets was supplemented with either lysozyme (100 mg/kg) or neomycin/oxotetracycline. After 14 days, not only did pigs fed the supplemented diets with either lysozyme or antibiotics gain weight faster and thus ended with 10% higher body weight, but also the small intestinal morphology was improved, as crypt depth was increased in both the jejunum and ileum, compared to the control diet (without supplementation of any kind). Also, the presence of Campylobacter, a pathogen causing intestinal infections, was detected in 27% of the control samples, compared to only 5 and 8% of samples from pigs fed with antibiotic-supplemented or lysozyme-supplemented diets, respectively [79,80]. In principle, lysozyme may not directly degrade the cell wall of the Gram-negative Campylobacter; however, lower detection of this pathogen may reflect enhanced gastrointestinal health of the animals fed with a lysozyme-supplemented diet. Similar beneficial results regarding body weight gain (WG) and final body weight were observed for 24-day old pigs fed for 28 days with a 100 mg/kg lysozyme-supplemented diet. It was suggested that the improved growth performance in the nursery pigs (10% greater than in pigs consuming control diets [p < 0.001]), may be due to improved histomorphology of the small intestine as well as a stronger nonspecific immune response [81,82]. Thus, there is evidence that direct lysozyme supplementation of diets effectively promotes growth, body weight, and intestinal health in young pigs, as traditional antibiotics do [66].

Lichtenberg et al. studied the effect on broilers of a microbial lysozyme (muramidase 007) from A. alcalophilum. A significant improvement was observed in WG (70.0 g/day), when the animals were fed with a muramidase 007 supplementation, compared to the diet without the enzyme (WG = 66.5 g/day); the overall feed conversion ratio (FCR) (g feed/g gain) was also significantly improved (1.505) with the enzyme-supplemented diet compared to the non-supplemented diet (1.547). Interestingly, dietary supplementation with muramidase 007 enhanced the number of lactobacilli without disturbing the rest of the functional microbiota in the caecum of broilers [83]. In a different study, Boroojeni et al. [84] reported that muramidase 007-supplemented diet for broiler chickens resulted in a linear increase in body WG, albeit a decrease in FCR (p ≤ 0.05) was observed. The results suggested that the inclusion of muramidase 007 in broiler diets increased the apparent ileal digestibility (AID) of key nutrients thus improving the growth performance. Abdel-Latif et al. [85] investigated the effects of the dietary lysozyme on the growth performance and the immune response of broiler chickens, at several dosages of 70, 90, and 120 g of lysozyme 10%® per ton of basal diet, for 5 weeks. The results revealed considerable amelioration in the growth performance and gut environment of the animals. A remarkable decrease of the harmful fecal Coliform and Clostridia and an increase (p < 0.05) of the beneficial Lactobacillus was observed in the lysozyme-supplemented groups, in comparison to control. Furthermore, the birds fed with 90 g of lysozyme 10%® per ton of basal diet had a significant increase (p < 0.01) in the glutathione peroxidase gene expression, which is considered to reflect the antioxidant status of the gut. Other works support the notion that lysozyme inclusion on broiler diets affects the composition of the microbiota; a 40 mg lysozyme/kg diet led to a decrease in intestinal colonization with C. perfringens and an improved intestinal barrier function, as well as growth performance, in broilers [86].

In aquaculture, the accumulation of antibiotics results in the development of resistant bacteria, thus the use of lysozyme instead of traditional antibiotics has been explored [57]. It has been demonstrated that lysozyme supplementation in fish feed can improve the digestibility and absorption of fish feed ingredients, increasing the growth parameters of aquacultural animals [87]. For instance, the effects of dietary lysozyme on growth performance and hematological indices of rainbow trout (Oncorhynchus mykiss) fingerlings were investigated; the results of this study showed that although there was no significant difference in the growth performance of fish fed with different levels of dietary lysozyme, the hematological indices such as white blood cell, red blood cell, and hematocrit were remarkably increased compared to control diet group [88]. In another work, Nakamura et al. investigated in carp, Cyprinus carpio L. the protective effect of lysozyme-galactomannan or lysozyme-palmitic acid conjugates. They reported that feeding for 8 days with lysozyme conjugates significantly reduced Gram-negative Edwardsiella tarda infection, and the survival rate was increased after supplementing the dietary lysozyme (after 6 days of cultivation with 30% for lysozyme-galactomannan conjugate-treated fish and 20% for lysozyme-palmitic acid conjugate-treated fish); in contrast, all control fish died within 3 days [89].

The conclusion that lysozyme improves growth performance and health indices was confirmed by these and other research works summarized in Table 4. The mechanisms leading to these beneficial effects are not well understood and could be due to several factors, including the antibacterial activity of lysozyme but also secondary effects of it, such as immumodulatory effects [90].

4 Polymer-based nanomaterials for lysozyme encapsulation

In general, because of safety, long-term dosing, and manufacturing costs, oral delivery of drug molecules, both small molecules and biomacromolecules, is usually preferable. However, oral delivery of active macromolecules such as peptide- and protein-based drugs is a challenge due to barriers in the GIT [91]. For instance, an intensive acidic environment in the stomach might alter the ionization of the peptide- and protein-based drugs causing a change in their structure and/or function. Moreover, the presence of digestive enzymes in the stomach can degrade most of these protein and peptide drugs. Furthermore, pancreatic proteolytic enzymes such as trypsin are found in the lumen of the small intestine [92,93]. Overcoming gastrointestinal barriers in humans is a priority for optimum delivery of proteins, and it can be important in animals as well, which in turn is a formidable challenge. This is exactly where a formulation of protein-based drugs through various tools and methods can address this challenge. Encapsulation of lysozyme may be one of the best methods to prevent degradation under the harsh conditions of the GIT. In this regard, several methods have been attempted, both using inorganic and organic matrixes. Most interestingly is the use of nanoscaled preparations, as size is a relevant physical property for cellular uptake, which influences the entrance of species and thus their potential to disturb cellular processes [94,95]. In the case of inorganic-based formulations, although positive results have been observed in terms of the antibacterial effect of the obtained material, including a synergistic effect of the inorganic component (for examples, see previous studies [96–99]), safety issues regarding its toxicity, especially in the case of metal-containing materials, may preclude its use as an additive in animal feed. Negative effects such as the generation of reactive oxygen species, decrease of cell viability, DNA damage, neurotoxicity, and other phenomena in human, animal, and plant cells have been reported [100–102] for nanoparticles (NPs) based on Ag, TiO₂, Au, Co, Al₂O₃, ZnO, Pt, Si, and SiO₂, to mention a few. There is a higher toxicity associated with the nanometric form of metal-based particles, when compared to the corresponding bulk form, which depends on the size, shape, surface chemistry, chemical composition, solubility, and aggregation behavior, among others, of the metal-based NP. The risk of toxicity is not fully elucidated, thus safe use of these materials in animals and humans is still under study to determine if the benefits compensate for the risks.

On the other hand, several synthetic and natural biopolymers have been shown to be biocompatible and nontoxic when included in nanoformulations. Polylactic acid, polyethylene glycol, dextran, pectin, cellulose, carrageenan, and alginate among others have been authorized by the FDA as inactive ingredients for inclusion in certain drug products due to their nontoxicity [103]. Moreover, there are several studies demonstrating the absence of toxicity of nanostructured biopolymers such as chitosan. For example, Sorasitthiyanukarn et al. [104] evaluated the cytotoxicity of chitosan/alginate NP loaded with curcumin diglutaric acid (CG), using three types of cancerous cells, MDA-MB-231, HepG2, and Caco-2. Empty NPs did not show any cytotoxicity, whereas free CG and CG-loaded chitosan/alginate NP showed noticeable cytotoxicity in all cell lines [104]. In another work, an in vivo toxicity study was performed by Aluani et al. [105], using repeated oral administration of empty and quercetin-loaded chitosan/alginate NP to male Winstar rats for 14 days. No mortality or changes in behavior and body weight was observed during the study, in all experimental groups [105].

In this review, we will focus on the use of nontoxic, polymer-based nanoformulations of lysozyme. Other lysozyme nanoformulations can be revised in the work of Sarkar et al. [106]. In the quest for developing efficient nanoformulations, multiple techniques are utilized to characterize polymeric NP. The critical parameters are size distribution, surface charge, and stability of the polymeric NP. Dynamic light scattering (DLS) can be used as a pivotal tool for providing information about the size distribution of NP, as well as the stability in solutions or suspensions [107]. Through DLS, the hydrodynamic diameter of the polymeric NP is accurately determined, providing crucial insights into their size homogeneity and dispersion [108]. Moreover, establishing the electrostatic interactions governing particle behavior is also helpful for evaluating stability. Therefore, electrophoretic light scattering is utilized to ascertain the zeta potential, or surface charge, of the polymeric NP. Zeta potential measurements offer valuable information regarding the potential for particle aggregation or dispersion in solution, guiding the optimization of formulation parameters [109]. The long-term stability of polymeric NP can be evaluated by monitoring changes in size and zeta potential over an extended duration [110,111]. This assessment offers insights into the robustness of the nanoformulations and their propensity for aggregation over time. By employing these characterization techniques tailored to polymeric NP, we can ensure a comprehensive understanding of their physicochemical properties.

Abu Abed et al. [112] conducted a study involving the formulation of polymeric nano-capsules using poly(d,l-lactic-co-caprolactone) (PLC) at varying monomer ratios. This was achieved through the double emulsion solvent evaporation method, employing a water-in-oil-in-water (W/O/W) emulsion approach. The primary objective was to explore their potential for encapsulating and releasing lysozyme in both simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The biological activity of the enclosed lysozyme was assessed by employing a method where polymeric NPs were disrupted using ethyl acetate, and the ability of the encapsulated lysozyme to lyse the cell wall of Micrococcus lysodeikticus was measured. Specifically, the calculation involved recording ΔA450 for the bacterial-lysozyme suspension over a 5 min interval. This study yielded spherical PLC nanostructures with an average size ranging from 325.3 to 865.3 nm. Within this system, the loading efficiency of lysozyme achieved 80%, a parameter notably influenced by the ratio of monomers. Notably, less than 10% of the encapsulated lysozyme was released in SGF, while the highest cumulative release percentage in SIF over 24 h was recorded at 73%. The sustained release phase was attributed to multiple mechanisms such as the weakening of particle structures due to a plasticizing effect, polymer degradation leading to matrix erosion, and the diffusion of the drug out of the polymer matrix due to factors like the formation of pores or water channels within the polymer shells as the medium penetrated the particles. Furthermore, the study highlighted that the encapsulated lysozyme retained a range of 39.69–97.42% of its original biological activity. The researchers found that biological activity significantly increased with higher amounts of trehalose (p-value < 0.011) and when lysozyme was encapsulated in a solid state (p-value < 0.003). The stability of lysozyme in the solid state was attributed to the limited hydrolysis rate resulting from the absence of water. The presence of water around lysozyme was recognized as a factor that could cleave amide bonds and potentially denature the protein during the preparation process. Trehalose was observed to stabilize proteins in solutions through a preferential hydration mechanism that reduces protein mobility, thereby maintaining the protein’s folded conformation.

Biopolymers have significant advantages over synthetic polymers for medical applications. Particularly polysaccharides are not only an abundant, environment-friendly resource, but they tend to form structured nanomaterials that are biocompatible and have been widely studied for different medical applications, from tissue engineering to drug delivery [113]. Chitosan is the most studied biopolymer to successfully encapsulate lysozyme while retaining the enzyme activity. Chitosan is a cationic biopolymer consisting of β-1,4-linked glucosamine and N-acetylglucosamine units (Figure 3). It is obtained by partial deacetylation of chitin, and due to its water solubility, non-toxicity, biodegradability, biocompatibility, and chemical modifiability, chitosan-based materials have gained huge attention over the past decade [114].

Figure 3

Summarized advantages of lysozyme as animal feed additive.

In this regard, Deng et al. prepared chitosan NPs through the ionic gelation method using tripolyphosphate (TPP) as a crosslinker for the encapsulation of lysozyme. To better optimize the encapsulation of lysozyme, the effect of various factors was investigated such as the molecular weight of chitosan and the concentration of chitosan, TPP, and lysozyme in the gelation reaction. Spherical shape chitosan NPs were obtained in the size range of 50–280 nm in diameter and lysozyme loading efficiency was up to 47.3%. The activity of lysozyme in all the samples was retained and was above 85% for that observed for soluble, free lysozyme [115].

Similarly, Wu et al. [116] reported a method of ionic gelation to form lysozyme-loaded chitosan NPs. Their results showed that upon lysozyme encapsulation, the NP size increased, and the pH of the gelation process affected the magnitude of this increase: at pH 4, NP size increased from 476.2 to 488.8 nm, whereas at pH 5 the size increased from 548.1 to 613.5 nm. The pH of gelation also influenced the encapsulation efficiency and lysozyme load, showing best results at pH 4 and an initial concentration of lysozyme of 1.25 mg/mL. Moreover, lysozyme-loaded chitosan NP increased the antibacterial activity against Gram-negative E. coli O₁₅₇ (26% increase) and Gram-positive Bacillus subtilis ACCC10242 (10% increase), compared to chitosan NP alone, measured as the inhibition zone diameter.

In another work, smaller NPs were also obtained through the ionotropic gelation technique of chitosan, generating NP with an average size of 150 nm [117]. Interestingly, the NPs were effective in maintaining the enzyme’s antibacterial activity toward S. epidermidis for up to 5 days, even though it was slowly released over 3 weeks in vitro. Moreover, the authors of this research demonstrated that lysozyme-NP sustained antibacterial activity beyond the intrinsic antibacterial activity of controls with either chitosan or lysozyme alone, resulting in about a 2-log reduction in viable S. epidermidis.

In a different approach, cellulose nanocrystals (CNC) were added to the medium during the formation of lysozyme-loaded chitosan NP through the ionotropic gelation technique, resulting in a material with both enhanced antibacterial properties as well as improved stability [118]. CNC, derived from sulfuric acid-treated microcrystalline cellulose, are materials with sulfate groups on the surface that in principle could favorably interact with the positively-charged chitosan. Two types of chitosan (low [LMW] and high molecular weight [HMW]) were utilized to generate the NP in the presence of CNC, yielding particles with different sizes (140 and 284 nm, respectively) albeit similar properties in terms of maximum encapsulation efficiency (59.38 and 51.23%, respectively) using 1.5 mg/mL of lysozyme, as well as a similar release of lysozyme over time (around 75% release at pH 7.4 after 12 h). However, an improvement in the antibacterial activity was observed for CNC-containing LMW NP. In this regard, the inhibition zone was larger when applying CNC-stabilized lysozyme-loaded LMW NP vs lysozyme-loaded LMW NP alone, with 13.29 vs 11.60 mm for E. coli and 15.29 vs 10.33 mm for Listeria innocua, respectively. The minimal inhibitory concentration of lysozyme alone (1.5 mg/mL), lysozyme-loaded LMW NP, and CNC-stabilized lysozyme-loaded LMW NP was 3.12, 0.624, and 0.312 mg/mL for E. coli and 0.78, 0.156, and 0.078 mg/mL for L. innocua, respectively. Interestingly, LMW chitosan NP alone showed an antibacterial effect, effectively preventing the growth in liquid culture of both tested bacteria. Probably the structuration of chitosan at the nanoscale aids in penetrating the cell membrane and may even disrupt the cellular function at other levels.

In conclusion, it is crucial to acknowledge that lysozyme is renowned for its bactericidal activity, and also that its integration with nanomaterials enhances its antibacterial activity. Notably, nanomaterials like chitosan NPs can immobilize lysozyme, thereby amplifying its antibacterial efficacy against a spectrum of bacteria, including those prevalent in livestock farming [119]. Mainly, two reasons could be envisioned for this phenomenon: first, the delivery to the intestinal tract of a higher concentration of active lysozyme due to protection conferred by polymers that are not easily degraded by the acidic conditions and/or enzymes present in the digestive system, mainly in the stomach. Second, a synergistic effect between nanostructured polymers and lysozyme. Both the catalytic and non-canonical activity of lysozyme could allow better adhesion and/or internalization of the NPs, which may have their own antibacterial activity once inside the cell, upon binding to specific targets.

5 Challenges and future research directions

Despite its evident health benefits for animals, several challenges must be overcome to reach a commercial application based on lysozyme. Perception issues could be an obstacle, so it is an important goal to have a favorable reception from animal breeders to use lysozyme instead of small antibiotics. The perception is highly influenced by economic aspects, so large-scale trials in animals to determine the relation of cost–benefit would be necessary. Most importantly, it is elementary to assess the safety of the prolonged use of lysozyme in animal diets. The main concern is the appearance of antibacterial resistance. The synergy of NP with lysozyme can elevate antimicrobial activity by disrupting bacterial membranes and inducing oxidative stress, thereby potentially instigating novel resistance mechanisms [120]. The pervasive use of antibiotics in livestock farming has been implicated in the rise of antibiotic-resistant bacteria, posing a substantial threat to public health [121]. Consequently, vigilant monitoring of the prolonged application of lysozyme-containing nanomaterials in livestock farming is de rigueur.

It is known that some bacteria are resistant to lysozyme, including human pathogens such as Haemophilus influenzae, Neisseria meningitidis, E. coli, S. pneumonia, K. pneumoniae, S. aureus, Neisseria gonorrhoeae, Enterococcus faecalis, and Proteus mirabilis [122–124]. These cases appear to be due to natural resistance, as opposed to acquired mechanisms. Resistance to lysozyme is based on the following mechanisms, although not all of them are present in all resistant bacteria [90,125]:

1. The production of capsular polysaccharides hinders access of lysozyme to the cell wall.

2. Alterations in the peptidoglycan structure and chemical nature reduce the efficiency of the catalytic hydrolysis of the peptidoglycan chains. For example, the N-deacetylation of NAG, O-acetylation, and N-glycolylation of NAM moieties of the peptidoglycan reduce the recognition of the substrate by the enzyme. Interestingly, this has been observed for lysozyme from chicken egg, but not for muramidases from other sources [126].

3. Certain modifications to the peptidoglycan change the bacterial surface charge or its permeability, thus inhibiting the non-canonical effect of lysozyme. For example, the amidation of aspartic acid residues in the peptide bridge or a larger degree of cross-linking of the peptidoglycan, respectively, can cause this effect.

Perhaps the most worrisome resistance mechanism is the expression of lysozyme-inhibiting molecules [90,127]. For example, a small group of Gram-negative bacteria such as E. coli and P. aeuroginosa produces a LMW protein, Ivy (inhibitor of vertebrate lysozyme), with high affinity to lysozyme from chicken egg (Ki = 1 nM, in vitro) [128,129]. Different, non-related proteins, termed PliC/MliC (periplasmic/membrane-bound lysozyme inhibitor of c-type lysozyme) and that are also produced by Gram-negative organisms such as Salmonella enteritidis, E. coli, P. aeuroginosa, and Aeromonas hydrophila, showed high affinity toward lysozyme (K _A = 10⁸–10¹⁰ M, [130,131]). Recently, a protein with structural but not sequence similarity (less than 20%) to PliC/MliC was reported in Neisseria spp. The adhesin protein complex (ACP) is exposed to the surface of the bacteria, and inhibited lysozyme action in vitro, while the growth of knock-out mutants was reduced by 3–9 fold [132]. These intricate and multifaceted resistance mechanisms have been documented across various bacterial species, including notable pathogens such as S. aureus and L. monocytogenes, and deserve further study [127,133].

Derived from these challenges, future research directions may be envisioned. As mentioned, research on the possible appearance of bacterial resistance to lysozyme when used in animal feed is essential. Along the same line, although proteins and natural polysaccharides are biodegradable, it is a good practice to perform a cradle to grave analysis for new products, to ensure that no undesirable damage to the environment or further problems could arise from the use of the proposed materials. Regarding the safety of lysozyme, it has been declared not toxic to humans and granted the status of “generally regarded as safe” by authorized organizations such as FAO, WHO, and the FDA [134,135]. No regulatory frameworks are available for the presence of lysozyme in animal feed, yet. This issue is very relevant and if lysozyme, either free or formulated in various fashions, is to be included in animal diets, its general safety will have to be assessed. The fact that it is safe for human consumption is a positive precedent [62].

Also, the subject of large-scale production of lysozyme at low cost is an area of research opportunity. To date, chicken eggs are the main source of commercial lysozyme. The average protein content in egg white is 11%, and lysozyme is approximately 3–4% of this, thus it is possible to obtain at least 3–4 g/L of lysozyme from this natural source. The cost could be lowered if modern biotechnology tools could be used to obtain recombinant, active lysozyme from heterologous systems such as yeast. Already some works have reported the production of recombinant lysozymes, for example from chicken eggs and human, in heterologous systems such as yeast, fungi, and bacteria [136]. Biotechnological processes could reduce the cost of lysozyme production, as well as ease the purification methods to obtain high-quality protein, without allergens. Although polysaccharides and other biopolymers, particularly those from biological origin sources, are already produced at large scale, nanoparticle production is still an emerging area. Reports about the technologies to produce biopolymeric NPs at industrial scale are very recent [137,138] and for this reason, we expect to see advances in the next years.

Finally, elucidating the mechanisms of the beneficial effects of lysozyme on animal health is relevant in improving or combine treatments. The study of the in vitro simulated gastric and/or intestinal environments and also the study of in vivo release of lysozyme, including the issues related to cell internalization of NP, and the effects on the microbiota, are research topics that should lead to a better understanding of the benefits of adding lysozyme to animal diet.

6 Conclusions

Lysozyme is a natural, protein-based antibiotic widely used in the food industry. Its beneficial effects on animal growth and health when fed as an additive have been demonstrated, as well as its potential to replace small-molecule based antibiotics in animal feed. Also, many possibilities to formulate lysozyme-containing biocompatible products are available, particularly taking advantage of synergistic effects when using nanopreparations. The advantages of the use of lysozyme are represented in Figure 3. More important, lysozyme-based products could replace the massive use of antibiotics, helping to alleviate the problem of micropollution in water bodies all over the world, which in turns may reduce the appearance of MDR pathogenic bacteria, a supreme challenge the entire world has to face to reduce mortality in the next decades. However, essential questions such as the potential appearance of AR when lysozyme is used as an animal food additive, as well as the mechanisms behind animal health improvement, remain to be answered before a widespread use of this or other enzyme-based alternative is allowed.