There is essentially a three-step model system of bacterial host lysis. This model is made up of three proteins: endolysin, holin, and spanin. These proteins act on the bacterial outer membrane, inner membrane, and peptidoglycan, and can cause host lysis. In Gram-negative hosts, there are two different processes for peptidoglycan degradation: (1) Holin-endolysin and (2) Pinholin SAR endolysin. Degradation of the peptidoglycan is in the preceding stage when holin makes small pores in the host inner membrane; lysis is then initiated by releasing endolysin inside the periplasm and depleting the peptidoglycan. In the final phase, lysis begins when the pinholin may induce membrane depolarization, activating the secreted SAR endolysin. Previously, it was thought that degradation of the first two membrane barriers was sufficient for the lysis of Gram-negative hosts, but current research indicates that a third functional class of lysis proteins, spanins, is now believed to be essential for outer membrane disruption. Spanins get their name from the fact that they form a protein bridge that connects both membranes. Most phages create a two-component spanin complex, which is made up of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) containing a coiled-coil periplasmic domain. With the N-terminal lipoprotein signal and a C-terminal transmembrane domain, some phages have a novel sort of spanin that spans the periplasm as a single molecule. Furthermore, the meshwork of the peptidoglycan inhibits spanin action, thus linking the spanin step to the first two processes mediated by holin and endolysin. These proteins can cause the bacterial host cell to lysis (Cahill and Young, 2019; Abdelrahman et al., 2021). Figure 1 illustrates the mechanism of action of endolysin.

The majority of endolysins have a modular structure, whereas endolysins acting on Gram-negative bacteria have a basic globular structure (Oliveira et al., 2018). Modular endolysins are differentiated by the presence of one or two (multi-domain) N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall-binding domain (CBD) by a short, flexible linker region. Phages infecting Gram-positive bacteria and mycobacteria both possess such a structure (Oliveira et al., 2018; Gondil et al., 2021). The N-terminal EAD of modular endolysins cleaves specific peptidoglycan bonds in the host bacterium’s murein layer, whereas the C-terminal CBD detects and attaches to various epitopes in the cell wall for proper binding of the EAD’s catalytic effects (Matamp and Bhat, 2019). The bacteriophage-derived endolysins of Gram-negative hosts can be organized in different ways, but the majority of Gram-negative endolysins contain a basic globular EAD domain without a CBD (Briers et al., 2007). The new investigation additionally found Gram-negative endolysins with globular structures, with one or two CBDs at the N-terminus and the EAD module at the end of the C-terminal region (Briers et al., 2007).

Endolysins are classified according to their location of cleavage position. The categories of these enzymes are lysozymes (N-acetylmuramidases), glycosidases (N-acetyl--d-glucosamidases), N-acetylmuramoyl-l-alanine amidases, and L-alanoyl-d-glutamate endopeptidases (Fenton et al., 2010; Abdelrahman et al., 2021). Phage-encoded cell wall hydrolases are a general word that could be used to describe these enzymes. Generally, endolysins consist of one of these four N-terminals linked to a different cell wall-binding domain (Murray et al., 2021).

Endolysin is mainly active in Gram-positive bacteria that lack a protective outer cell membrane. The existence of an outer membrane in Gram-negative bacteria limits but does not entirely prevent the function of endolysin (Murray et al., 2021). Endolysin often has a broader range of specificity than phages, and their specificity is typically at the genus or species level. Endolysins’ selectivity for bacterial targets is due to their cell wall-binding domains, which detect and bind receptors independently to a specific substrate within the target cell wall. Due to this reason, Gram-negative lysins have a wider range of targets, while Gram-positive targeted lysins often have a narrower host range (Ajuebor et al., 2016). Endolysins with broad-spectrum activity against Gram-positive bacteria have been reported. A pneumococcal bacteriophage-derived lytic endolysin Pal can eradicate 15 prevalent pneumococcal serotypes, even those with a high level of penicillin resistance (Loeffler et al., 2001). The endolysin phi11 shows potent antimicrobial activities against various Staphylococcus strains including S. aurues, S. epidermidis, S. hyicus, S. simulans, S. xylosus and S. wraneri (Donovan et al., 2006). In many other studies, the endolysins’ efficacy against Gram-positive bacteria has been reported including ClyR, ClyJ, ClyV, ClyC, SAL200, LysK, and Cpl-1 (Yang et al., 2015a; Seijsing et al., 2018; Bae et al., 2019; Gondil et al., 2020a; Huang et al., 2020; Yang H. et al., 2020; Li X. et al., 2021). Some of the examples of endolysin showed broad-spectrum activity against Gram-negative bacteria. The endolysins LysAB54, LysP53 and LysPA26 showed antibacterial activity against multidrug-resistant Gram-negative bacteria such as A. baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli (Guo et al., 2017; Khan et al., 2021; Li C. et al., 2021). The reported endolysin Abtn-4 and LysSS has a wide range of bactericidal activity against a wide range of Gram-positive and Gram-negative bacteria, including E. coli, A. abauminii, P. aeruginosa, K. pneumoniae, Salmonella spp. and S. aureus (Kim et al., 2020; Yuan et al., 2021).

Endolysins are regarded as persistent therapeutic agents in terms of bacterial resistance development. Their specificity for a bacterial species or genus gives them several advantages over broad-spectrum antibiotics. The co-evolution of phages with their host bacterium has brought numerous benefits, including conserved binding ability and activity, as well as a rare possibility of mutation in binding sites, making resistance to endolysins a remarkable finding. Because the majority of antimicrobial resistance mechanisms are within the cell, the extracellular application of endolysins and the presence of target peptidoglycan on the cell’s outer surface prevent the development of resistance to endolysin (Gondil et al., 2020b). The repeated exposure of Streptococcus pneumoniae to endolysin Pal at a low concentration shows no resistance (Loeffler et al., 2001). Experiments were performed and attempted to bring resistance in Bacillus spp. against endolysin PlyG and methicillin-resistant S. aureus against endolysin ClyS. Still, no resistance occurred in both bacteria against their corresponding endolysins, while antibiotic resistance occurred in these bacterial strains using the same methodology (Schuch et al., 2002; Pastagia et al., 2011). There is no published report on the resistance of bacteria to endolysin.

To protect its host from attacking pathogens, the normal microbiota develop a physical barrier (Cash et al., 2006). Any disruption in the natural microbiome can lead to significant disease (Wang et al., 2017). Antibiotics facilitate human health and life span; yet, broad-spectrum antibiotics disturb the existing microbiota, resulting in dysbiosis and adverse effects (Keeney et al., 2014). Endolysins, compared to typical broad-spectrum antibiotics, can target selected bacterial species with minimal adverse effects on the surrounding microbiome, and the absence of bacterial resistance mechanisms has also been reported as a benefit of the use of endolysin. Because of these characteristics, endolysins have been allocated to a new class of antimicrobial agents known as enzybiotics (Murray et al., 2021). PlyV12, a bacteriophage-derived endolysin showed a strong lytic activity against the vancomycin-resistant strains of Enterococcus faecalis and E. faecium. Endolysin causes less disruption of the normal microbiota because they do not transfer resistance genes or bacterial toxins that harm the colonizing bacteria of mucous membranes (Rahimzadeh et al., 2018). They can stimulate the immune system without neutralizing or inhibiting antibacterial action. As a result, they can be used to treat systemic infections (Fischetti, 2008). Imanishi et al. (2019) investigated the therapeutic potential of a recombinant S25-3 against S. aureus. They found out, firstly, that the recombinant endolysin S25-3 was effective against S. aureus. Secondly, in an experimental mouse model of impetigo, topical administration of recombinant S25-3 endolysin reduced the number of intraepidermal Staphylococci. Thirdly, in the same mice, treatment with recombinant S25-3 endolysin boosted the diversity of the skin microbiota. The Pal in-vivo analysis showed that microbial species commonly present in the human oropharynx were not affected by the Pal endolysin (Loeffler et al., 2001). These findings showed that endolysin has no adverse effect on the human microbiota.

Bacteriophages are part of the human microbiome. Hence bacteriophage-derived endolysin is unlikely to harm human health (Navarro and Muniesa, 2017; Kirsch et al., 2021). Endolysins can surpass the immune system due to their fast action and specific receptors of the CBD on its target host (Jado et al., 2003). Still, their eventual acceptance as antimicrobial agents for the general public heavily depends on their immunogenicity, how the body’s immune system will respond to them, and their safety assessment on human health (Abdelrahman et al., 2021). In this regard, several studies show endolysins’ safety assessment. The safety and toxicity of two pneumococcal endolysins, Pal and Cpl-1 were investigated in human macrophages and pharyngeal cells. Similarly, no physical or behavioral changes were observed in mice injected with 15 mg/kg of each endolysin. IgG levels in mice exposed to the Streptococcus pneumoniae endolysins Cpl-1 and Pal increased. However, IgE levels remained low, suggesting a low probability of hypersensitivity or allergic reactions. Additionally, no adverse health consequences in mice with increased levels of pro-inflammatory cytokines or complement activation were seen, suggesting that these endolysins had good safety and toxicity profiles. The Pal and Cpl-1’s endolysin safety and toxicity characteristics justify the safe use of endolysin (Harhala et al., 2018). PlyCD1-174 is effective against Clostridium difficile spores infecting the large intestine in the presence of its intestinal contents. The PlyCD1-174 was injected into the colons of C. difficile-infected mice and 400 μg of endolysin delivered via the colon had no abnormal adverse effects, such as diarrhea or mice weight loss (Wang et al., 2015). In rodent single-and repeated-dose toxicity trials, intravenously administered SAL200 revealed no adverse effect. There were no adverse findings in the dog repeated-dose toxicity test, except temporary abnormal clinical symptoms found in certain dogs when daily injection of SAL200 was continued for more than one week. All aberrant findings found in these safety review investigations were minor and were rapidly recovered and no animal deaths were reported in the safety assessments (Jun et al., 2014). In a clinical trial, healthy male volunteers were administered the endolysin SAL200, and the results showed good endolysin tolerance in human subjects. Therefore, endolysins have an excellent safety profile and revealed no side effects like fever, stomach cramps, or diarrhea in preclinical studies conducted using animal models, demonstrating their safety aim (Jun et al., 2017). The safety and immunogenicity efficacy of endolysin ClyR was investigated in a systemic Streptococcus agalactiae infection mouse model. The ClyR rescued the mice and had no harmful effects on the mice (Yang et al., 2015a). The endolysin MV-L safety activity is evaluated in the mice. The mice who received a single high dosage of endolysin followed by a repeat dose did not show any side effects or a change in their survival rate (Rashel et al., 2007). The endolysin PlyC, immunogenicity, and allergic reactions were tested in mice and human serum samples from healthy volunteers. Mice were challenged with PlyC, and humans lacked PlyC-specific IgE. PlyC was immunogenic but did not cause hypersensitivity. Furthermore, adverse effects, particularly those indicating hypersensitivity, were closely monitored, and no adverse reactions were recorded. These findings significantly support PlyC’s safety as a possible therapeutic enzyme that can be applied to treat severe bacterial infections (Harhala et al., 2022). The endolysin LysGH15 is tested for the influence of endolysin inactivation by antibodies. LysGH15-specific antibodies did not impact LysGH15’s ability to kill MRSA in vitro or in vivo (Zhang et al., 2016). The high safety of endolysins could be because endolysin is very specific for distinctive and conserved structures of bacterial cell wall peptidoglycan or polysaccharides that are not present in mammalian cells (Nau and Eiffert, 2002; Nelson et al., 2012). Although the findings of safety studies to date support the claim of endolysins as a potential safe antibacterial agent. More research in this area is needed to confirm that different classes of endolysin do not pose a significant concern to the host (Murray et al., 2021).

Biofilms are microorganism communities that are sessile and embedded in a self-produced extracellular environment, being the most widespread bacterial lifestyle (Qian et al., 2022). The well-organized structure of these multilayer colonies guards bacterial cells against external threats, improving their ability to tolerate antibiotic or disinfectant treatment. These colonies are identified by enhanced antibiotic resistance in both hospitals and foods. They are a persistent cause of re-infection and re-contamination of foods (Abee et al., 2011; Fernández et al., 2018). Bacterial biofilm formation is a defense mechanism that bacteria use to deal with unfavorable conditions such as food scarcity or toxic antimicrobial dosage. Antimicrobial drugs often have a limited effect on biofilms, which is hypothesized to be owing to several biofilm-specific features. Low growth rates hamper antibiotics, and evidence showed that sessile bacteria in biofilms have a different metabolic state. Antibiotics’ diffusion velocity is also restricted within biofilms. Reduced antibiotic penetration increases the antibiotic concentration in the biofilm’s deeper layers, allowing biofilm cells to adapt to the antibiotic through stress-induced metabolic and transcriptional changes (Srinivasan et al., 2021). Because of their considerable resistance, the S. aureus biofilms are recognized as a serious concern in the food industry. The reported endolysins LysCSA13, ϕ11, Lys109, LysP108, XZ.700 and LysK did the eradication of bacterial cells of the S. aureus and eliminated its biofilms from different surfaces, including polystyrene, glass, and stainless steel. These endolysins can be used as a potential biocontrol agent in the different food processing sectors (Sass and Bierbaum, 2007; Schmelcher et al., 2015; Cha et al., 2019; Kuiper et al., 2021; Lu et al., 2021; Son et al., 2021).

The L. monocytogenes and Salmonella are also increasingly pressing issues for food industries because of their biofilm formation. The recombinant endolysins PlyLM and 293-amidase showed anti-biofilm activity against L. monocytogenes while the endolysin Lys68 showed a synergistic biofilm-reducing activity against the salmonella in combination with malic or citric acid. Lys68 has good thermo-stability properties so it can be an alternative antibacterial protein against Gram-negative pathogens in the food industry (Simmons et al., 2012; Oliveira et al., 2014; Pennone et al., 2019).

Antibiotic misuse and overuse have resulted in a significant emergence of resistant bacteria in meat-producing animals, particularly cattle, poultry, and pigs, which have been identified as crucial carriers of antibiotic-resistant bacteria and their particular genes, which can transfer to humans through direct contact or indirectly through foods (Marshall and Levy, 2011). In response to increasing concern over antibiotic misuse in food-producing animals, many large food companies have agreed to eliminate antibiotic use and improve control over alternative treatments. This creates complex scenarios for the animal-husbandry sector, which can be overcome using bacteriophage-derived endolysins (Love et al., 2018). The poultry market has faced severe problems due to Clostridium perfringens, which can infect 95% of poultry (Timbermont et al., 2011). The endolysin phiSM101 showed a wide range of bactericidal activity against C. perfringens (Tamai et al., 2014). Salmonella can cause widespread disease in chickens, and it causes economic losses in the poultry business (Wernicki et al., 2017). The endolysin LysSE24 derived from the Salmonella bacteriophage LPSE1, showed antibacterial activity against the Salmonella strains (Ding et al., 2020).

Meat is a significant carrier of foodborne infections (Bantawa et al., 2018). The endolysin LysSA11 showed a 3-log CFU/mL of methicillin-resistant S. aureus (MRSA) bacterial reduction in ham meat at a refrigerator temperature of 4°C (Chang et al., 2017a). The endolysin Trx-SA1 effectively reduce S. aureus in the cow udders. Trx-SA1 can be used as an alternate bio-preservative to minimize S. aureus contamination during food storage (Fan et al., 2016).

Dairy-borne pathogens, such as L. monocytogenes, S. aureus, Salmonella, and Shiga toxin-producing E. coli (STEC), should be controlled in the dairy production industry. The lysis activities of endolysins have been investigated particularly in dairy products (Lee et al., 2022). S. aureus has been effectively eliminated by several endolysins in milk (Obeso et al., 2008; García et al., 2010; Chang et al., 2017a). The endolysins LysH5, LysSA97 and LysSA11 were tested in milk which showed strong bactericidal activity against S. aureus. LysH5 revealed a synergistic bactericidal activity in pasteurized milk when used in combination with nisin. These antimicrobial proteins can be effective milk preservative candidates (Obeso et al., 2008; García et al., 2010; Chang et al., 2017a,b). The C. tyrobutyricum has been linked to the late blowing of hard and semihard cheese. Even minute quantities of heat-resistant spores can result in food spoilage during pasteurization, processing, and canning procedures. The C. tyrobutyricum phage ϕCTP1 endolysin Ctp1L is an antibacterial protein that showed activity in milk and prevents late blowing during cheese manufacturing (Mayer et al., 2010). Listeria contamination must be eliminated when making fresh cheese since its delicate taste and textures are inconsistent with several antimicrobial treatments and chemicals commonly used in other foodstuffs. The endolysin PlyP100 can potentially prevent the spread of L. monocytogenes in fresh cheese (Van Tassell et al., 2017). The endolysins LysZ5, ply511, and ply118 lysis the L. monocytogenes in milk. LysZ5 has good host specificity and host lysis activity at refrigerator conditions. ply511 and ply118 showed broad-spectrum activity and had high thermal resistance (Zhang et al., 2012; Schmelcher et al., 2012c). The endolysin ply100 showed synergistic bactericidal activity in combination with nisin against the Listeria in the Queso Fresco cheese (Ibarra-Sánchez et al., 2018). Endolysins are also tested against foodborne Listeria in milk, soy milk and Queso Fresco cheese (Zhang et al., 2012; Schmelcher et al., 2012c; Ibarra-Sánchez et al., 2018).

To reduce and prevent the antibiotic resistance pathogenic bacteria in vegetables, endolysin has also been tested in vegetable products. Several endolysins have been proposed as possible antibacterial bio-preservative agents for vegetable products against L. monocytogenes in iceberg lettuce (Schmelcher et al., 2012a). The endolysin LysP53 can lysis Salmonella enteritidis on fresh romaine lettuce and can be employed as a biocontrol agent to lower bacterial burdens in fresh vegetable food due to its good activities against a wide range of bacteria, thermal stability, and safety (Li et al., 2023). The endolysin cpp-lys greatly lysis the bacteria C. perfringens which produced various toxins. The activity is checked and confirmed on lettuce. The results of this investigation showed that endolysin cpp-lys has potential uses in the food industry for the control of C. perfringens (Zhao et al., 2023). LysWL59 and LysWL60, are potential bacteriophage LPST10-derived endolysins, which revealed extensive lytic activity and diminished viable S. typhimurium on lettuce (Liu et al., 2019).

To ensure food safety, sensitive and specific diagnostic protocols that accurately and rapidly detect pathogenic bacteria in food are essential (Hagens and Loessner, 2007). A useful tool for the detection of a particular food-borne bacteria is CBD which has been fluorescently tagged (Loessner et al., 2002). CBDs derived from bacteriophage-derived endolysins are excellent choices for making use of novel detection tools due to their high degree of specificity and binding affinity. CBDs from Listeria phage endolysins are a prime example, as their binding affinity to the target cells closely resembles or exceeds antigen–antibody interactions (Loessner et al., 2002; Schmelcher et al., 2010). Furthermore, CBDs have highly specific yet varied binding spectra, ranging from the level of the genus to the level of the serovar or even the level of the strain, providing new opportunities for the detection, immobilization, and differentiation of cells (Schmelcher et al., 2010). The endolysin LysCPAS15 inhibited the bacterial cells by up to a 3-log reduction, and enhanced green fluorescent protein EGFP-fused CBD protein (EGFP-LysCPAS15_CBD1) detects C. perfringens (Cho et al., 2021). A novel method for detecting S. aureus in milk is developed by combining immunomagnetic separation with the enzyme-linked CBD of endolysin plyV12, which binds S. aureus with high affinity, tested in the contaminated milk. On the cell surface of S. aureus, there are millions of binding sites for CBD attachment, which significantly improves the detection sensitivity. This method could be used to detect S. aureus in different food samples due to its simplicity, fast detection, and high sensitivity (Yu et al., 2016). Fused CBDs were developed to target and detect other bacterial pathogens, such as B. cereus and L. monocytogenes (Ahmed et al., 2007; Eugster et al., 2011; Kong and Ryu, 2015). However, due to the outer membrane barrier, Gram-negative food-borne pathogens cannot be detected by CBD, which is a significant drawback. CBD cocktails can recognize numerous food-borne bacterial pathogens that are present in a food sample at once by using various colored fluorescent tags. For instance, a multiplex decorating with CBDs allowed the identification of various serovar groups of Listeria. The CBD-P35 and CBD500 were able to recognize various Listeria strains in both milk and camembert cheese samples when they were labeled with different fluorescent markers including RedStar and GFP (green fluorescent protein) (Schmelcher et al., 2010). Due to high specificity and binding activity for pathogenic bacteria, CBD is a perfect option to replace antibodies in the rapid detection of bacterial pathogens (Bai et al., 2016). Even though there has been tremendous advancement in the use of endolysins or their CBDs for detecting pathogenic bacteria in food, future studies should focus on further investigation of endolysins or their CBDs into the quickly developing biosensor technology (Schmelcher and Loessner, 2016).

Salmonella is usually recognized as one of the most significant and widespread foodborne pathogens in the entire world. Salmonella has over 2,000 serotypes, some of which can cause human enteric infections, also known as salmonellosis (Majowicz et al., 2010). Salmonella nontyphoidal, which causes food poisoning outbreaks, is China’s second most common foodborne pathogen (Fu et al., 2019). LysSTG2, En4 and SPN9CC bacteriophage-derived endolysins showed bactericidal activity against S. typhimurium. The LysSTG2 (100 μg/mL) and slightly acidic hypochlorous water (40 mg/L of chlorine) eliminated more than 99% of the S. typhimurium. En4 showed a reduction of S. typhimurium 1.0–1.6 log CFU/g in frozen and thawed raw chicken meat with the addition of 0.1% NaHCO3. These results suggested that using endolysin together with permibilizers could be a novel and effective way to combat S. typhimurium in the food industry (Lim et al., 2014; Zhang et al., 2021; Abhisingha et al., 2023). Figure 2 describes the different applications of endolysin.

Escherichia coli O157: H7 is a foodborne pathogen that causes a wide range of health issues in humans, including lethargy, nausea, bloody diarrhea, and, in extreme cases, death (Alhadlaq et al., 2023). The fresh vegetables are an essential part of a healthy diet. However, the contamination of salad greens with Escherichia coli O157: H7 has resulted in serious illness and significant economic losses (Xu et al., 2021). The endolysins PlyEc2 and RLysJN01 showed bactericidal activities against the E. coli O157:H7 on lettuce and did not affect the color or taste quality of the lettuce (Xu et al., 2021; Shen et al., 2022).

Staphylococcus aureus is the most common cause of foodborne illness around the globe (Kadariya et al., 2014). MRSA has been a considerable concern to public health worldwide. During the previous decade, an extensive spread of MRSA with cattle origin has been detected along the farm-to-fork food chain (Köck et al., 2014). One potential control technique is purified endolysins to serve as bio-preservatives in food products to control S. aureus food poisoning. The endolysins Lys109 inhibited S. aureus and MRSA in milk and bacon foods and has promising lysis activity on planktonic bacteria and biofilms (Son et al., 2021). CHAPSH3b (which includes the endopeptidase domain of HydH5 and the cell wall targeting domain of lysostaphin) has been reported which eliminate S. aureus in milk to undetectable levels and retained significant activity in raw milk, after pasteurization, and during storage at 4°C. For these reasons, CHAPSH3b has been proposed as a possible bio-preservative component for milk storage (Rodríguez-Rubio et al., 2013).

Listeria monocytogenes is a bacterium found in ready-to-eat foods such as meats, mixed salads, dairy, and vegetables (Zhu et al., 2017; Desai et al., 2019). L. monocytogenes is an infectious pathogen that can transfer to humans through food contamination. It can cause severe infections with possibly deadly outcomes in vulnerable groups like newborns, the old, pregnant women, and immunocompromised people (Vázquez-Boland et al., 2001; de Noordhout et al., 2014). Recently, researchers have studied, examined, and reported endolysin as a natural biocontrol agent against the foodborne pathogen L. monocytogenes. The endolysin PHA_BNPs showed prominent activity against L. monocytogenes (Stone et al., 2022). Fresh Hispanic cheese, known as queso fresco, is frequently linked to outbreaks of listeriosis in the US and human foodborne diseases caused by L. monocytogenes. The endolysins PlyP40 and PlyPSA prevented and eradicated L. monocytogenes contamination in queso fresco at cold storage for 28 days. These endolysins showed activity at low temperatures, pH, and salinity (Holle and Miller, 2021). The heat-stable L. monocytogenes endolysins, PlyP35, Ply511, and Ply118, is a fascinating finding, which shows lysis activity after 30 min of heating to 90°C. Ply511 and Ply118 decreased the amount of live L. monocytogenes when added to artificially spiked food items including iceberg lettuce and whole cow milk (Schmelcher et al., 2012a). These stable heat endolysin can be used in heat-treated food products like pasteurized milk (Schmelcher et al., 2012c). The endolysin LysZ5 from the L. monocytogenes phage FWLLm3 specifically reduced host growth up to 4 log CFU in soya milk in 3 h at 4°C, indicating that LysZ5 has excellent host specificity and host lysis activity at the refrigerator temperature (Zhang et al., 2012). Ply500, a listericidal peptidase, revealed a broad activity range across the genus Listeria (Schmelcher et al., 2012c).

Clostridium perfringens is a Gram-positive, anaerobic, and rod-shaped bacteria that can cause food poisoning through its enterotoxin (Baldassi, 2005). Fast detection and treatment of C. perfringens infection are essential for maintaining food safety (Cho et al., 2021). It has been reported that C. perfringens frequently contaminates milk (Mcauley et al., 2014). The endolysin LysCPAS15, Psa, Psm, PlyCP10 and PlyCP41 showed lytic activity against C. perfringens and as natural antibacterial proteins for preventing the spread and contamination in milk, meat and other food products. LysCPAS15 was tested in milk which reduced the bacterial load in milk. The PlyCP10 and PlyCP41 inhibited the growth of seventy-five C. perfringens strains isolated from pigs, chickens, and cows (Swift et al., 2018; Cho et al., 2021; Sekiya et al., 2021). Clostridium botulinum is a Gram-positive, anaerobic bacterium that produces spore’s botulinum neurotoxin (BoNT). BoNT causes botulism, a potentially fatal flaccid paralysis, in humans and animals. The germination and development of C. botulinum spores in food can cause different infections in humans and animals (Zhang et al., 2020). The endolysin CBO1751 efficiently lyses C. botulinum and also eliminates neurotoxin production. The antibacterial endolysin CBO1751 against C. botulinum, is a novel anti-botulinum protein for non-thermal applications in food and agriculture (Zhang et al., 2020). Clostridium sporogenes are spoilage bacteria that can cause food spoilage and rotting problems (Mayer et al., 2012). The endolysin CS74L showed lysis activity against the 16 tested strains of C. sporogenes (Mayer et al., 2012).

The main pathogen involved in seafood-borne bacterial infections is Vibrio parahaemolyticus. V. parahaemolyticus is a gram-negative bacterium inhabiting sea foods, including fish, shrimp and oysters (Letchumanan et al., 2014). The V. parahaemolyticus forms biofilms commonly in food manufacturing environments, contaminating food manufacturing machinery and products (Lopatek et al., 2018). V. parahaemolyticus biofilm production can increase resistance to antibacterial drugs commonly used in food, raising the chance of pathogen infections (Ning et al., 2021). Food contaminated with V. parahaemolyticus can cause stomach pain, nausea and vomiting, acute dysentery, and bloodstream infections (Baker-Austin et al., 2017). Additionally, due to antibiotic abuse, V. parahaemolyticus antibiotic resistance is becoming a severe concern to consumers (Shaw et al., 2014). Ninety V. parahaemolyticus strains from the main seafood in the China provinces of Shandong, Hebei, and Liaoning near the Bohai and Yellow Seas were recently examined for antibiotic resistance. Almost half of the isolates had at least three drug resistances, and more than 80% were resistant to ampicillin and cephazolin (Jiang et al., 2019). The demand for alternate antimicrobials to manage V. parahaemolyticus and associated biofilms may have recently increased (Ning et al., 2021). Endolysins are prospective substitutes for conventional antibiotics in the fight against V. parahaemolyticus and its biofilms (Matamp and Bhat, 2019). The recombinant endolysins Lysqdvp001, LysVPMS1 and LysVPp1 inhibited the growth of V. parahaemolyticus. Lysqdvpoo1 and poly-lysine showed robust synergistic antibacterial activity, so these two can be used in combination to tackle V. parahaemolyticus and its biofilms in the food sector (Wang W. et al., 2016; Li et al., 2018; Zermeño-Cervantes et al., 2018; Ning et al., 2021).

Campylobacter jejuni is the most common foodborne bacteria which can cause gastroenteritis worldwide and poses a severe risk to public health and food safety (Moffatt et al., 2016). The endolysin Cj1 and Cj5 can eradicate and control the C. jejuni in chicken food models in a refrigerated 5°C (Zampara et al., 2021).

Figure 3 defines the efficacy of endolysin as an antibacterial agent against foodborne illness caused by foodborne bacterial pathogens. Table 1 summarizes the use of endolysins in different food products, while Table 2 describes the efficacy of endolysin against different pathogenic bacteria.

The stability of endolysins used in the food is a main difficulty and concern. On a laboratory scale, natural and engineered endolysins have shown remarkable antibacterial activity. However, given that food is a massively complex of foodstuffs with distinct factors, such as ion concentration, acidity, salt concentration, temperatures, and physical properties, there might be a significant difference in their antimicrobial effectiveness in dietary settings compared to in vitro used (Shannon et al., 2020). The reported endolysins, LysAB54 and PlyA, showed a loss of bactericidal activity in the milk. These may be due to environmental factors like salt and different protein interactions, which can neutralize the endolysins (Yang et al., 2015b; Khan et al., 2021). Additionally, research into the structure of individual domains in endolysins, the location and characteristics of linker sections, the distribution of charges, and the use of cofactors can shed light on how endolysins interact with certain meals (Shannon et al., 2020). Novel delivery ways, such as encapsulating the endolysins into some nano-vesicles, may be attempted to synthesize Gram-negative endolysins for food safety (Gondil and Chhibber, 2021; Lee et al., 2022). At the same time, we suggest that advanced computational and structural analysis ought to serve as the foundation for endolysin designs that are more logically planned and can enhance antibacterial activity in particular foodstuffs.

During the stationary phase, the bacteria undergo several changes, including spherical cell growth, a hard cell envelope, intense cross-linking of the cell wall, a reduction in membrane fluidity, and the activation of the robust response mechanism to help the cells escape from the disruption (Jaishankar and Srivastava, 2017). Due to this reason, bacterial growth phases have a big effect on the activity of endolysin and endolysins show less activity against the stationary phase of bacteria. Endolysin can be combined with other antibacterial agents to achieve a synergistic effect and improve lysis activity. The engineering of endolysin with the outer membrane-permeabilizing polypeptide, chitosan nanoparticle, and other LPS disrupter molecules including EDTA, and citric acid (Murray et al., 2021; Kocot et al., 2023). Also, we suggest the combination of different endolysins can be used in synergism to enhance the activity of endolysin against the stationary phase of bacteria.

It has been discovered that several endolysins function as broad-spectrum antibacterial agents against Gram-positive bacteria (Yang et al., 2015a; Huang et al., 2020; Yang H. et al., 2020). Contrarily, a protective bacterial outer membrane renders most endolysins ineffective against Gram-negative bacteria (Abdelrahman et al., 2021; Khan et al., 2021). Recently, bacteriophage and endolysin encapsulation methods have been reported (Loh et al., 2021). Developing novel phage and endolysin delivery methods and their application are alternative strategies to overcome endolysins’ challenges and improve endolysins’ efficiency (Elkhoury et al., 2020). Therefore, through this strategy, the endolysins can interact with the peptidoglycan layer directly (Oliveira et al., 2012; Deshmukh et al., 2021). Moreover, Yves Briers described novel ways to disrupt the Gram-negative bacterial membrane: (1) physical or chemical methods to disrupt the outer membrane’s integrity; and (2) protein engineering to provide endolysins with the tools needed to overcome the outer membrane (Gutiérrez and Briers, 2021). Another method for combating Gram-negative pathogens is to fuse endolysin with a peptide with outer membrane-permeabilizing features to the endolysin. By attaching the peptide cecropin A to LysMK34 to its N terminus via a linker consisting of three Ala-Gly repeats, resulting in a genetically engineered LysMK34 (eLysMK34). Compared to the parental endolysin, the modified engineered endolysin shows improved antibacterial activity (Abdelkader et al., 2022). To boost outer membrane permeability and increase activity against Gram-negative bacteria, use endolysin in combination with antibiotics. LNT113, an engineered endolysin, was evaluated for synergistic effects with standard-of-care antibiotics. The LNT113 showed a synergistic activity with antibiotics (Hong et al., 2022). Hurdle technology: The use of hurdle technology provides a strategy for enhancing the efficiency of endolysins against Gram-negative bacteria. The makes use of multiple methods combined to eradicate bacteria and ensure the microbiological safety of food. In the case of endolysin, the procedure requires the use of another treatment first, which is to break cell membranes, including the outer membrane allowing the lysin to interact with the target petidoglycan (Oliveira et al., 2012; Mukhopadhyay and Gorris, 2014). The further strategies to increase the bactericidal activity of endolysin against gram-negative bacteria are the development of engineered endolysin. The methods of engineering of endolysin include the development of Lysocins, Innolysins, Lysin-based dendrimers, In silico search for antimicrobial peptides, Liposome-mediated lysin delivery, encapsulation in alginate-chitosan nanoparticles (Kocot et al., 2023).

Endolysins can eradicate Gram-positive bacteria has been proven in food matrices and specific food products, which strongly suggests that they could be used to fight foodborne pathogens in the food industry. However, the necessity for their large-scale production, purifying procedure, and scalability, which may include expensive costs, limits the use of endolysin in food companies (Xu, 2021). We should carefully assess the safety, stability, and manufacturing costs of engineered endolysins before using them in the food sector. Therefore, to address the remaining issues with endolysin, ongoing research into related issues is necessary due to the high needs of the food sector (Lee et al., 2022). Figure 4 shows the endolysin purification process.

Endolysins’ antibacterial activity, CBD dependency, and host specificity can all be altered by the truncation or deletion of a domain (Cheng and Fischetti, 2007). The Streptococcus endolysin PlyGBS were randomly mutated. The mutated endolysin had about 20-fold higher lytic activity than full-length PlyGBS. Horgan et al. (2009) synthesized several variants of the Staphylococcus LysK endolysin and reported that CHAPk (a truncated LysK-engineered endolysin) had better lytic activity against staphylococcal strains in vitro than the natural. Low et al. (2011) revealed that eliminating CBD from several endolysins improved their enzymatic activity. They explained this phenomenon in the context of an EAD domain’s CBD dependability being closely linked to its charge. Because lipoteichoic acids are negatively charged in the cell wall, changing the net charge of the catalytic domain from negative to positive may result in enhanced interaction between the catalytic domain and lipoteichoic acids.

The direct mutation was used further to improve the endolysins’ lytic activity and thermostability. Direct mutation in conserved sites involved in hydrogen bond networking at the backside of the amidase domain’s catalytic sites resulted in higher lytic activity of mutant CD27L endolysin against multiple L. monocytogenes serotypes (Mayer et al., 2011). Díez-Martínez et al. (2013) synthesized Cpl-7S, a synthetic endolysin, by swapping 15 amino acids into CBD. The alteration enhanced CBD’s net charge, resulting in improved lytic activity. For the changeable sites within the LysF1 fold, Love et al. (2021) altered a single residue in a non-conserved hydrophobic core to increase its heat stability. Using site-directed mutagenesis based on protein structure may be an appropriate approach for enhancing endolysins’ functions.

Endolysin domains can act independently, they can be replaced or recombined with domains from other endolysins to develop chimeric enzymes with desirable features. Indeed, multiple studies have shown that these tactics are effective in enhancing catalytic efficiency (Mao et al., 2013; Yang et al., 2014b; Díez-Martínez et al., 2015). And increasing host specificity through CBD shifting or addition (Yoong et al., 2004; Becker et al., 2009; Zhou et al., 2017). Fusion endolysins with improved lytic activity and thermostability have been effectively tested on milk and poultry (Rodríguez-Rubio et al., 2012). Rodrguez-Rubio et al. reported that the fusion protein CHAPSH3b which is composed of the CHAP domain of HydH5 and the SH3b domain of lysostaphin eradicated S. aureus in pasteurized milk to undetectable numbers after 15 min of treatment at a relatively low concentration of 1 μM (Rodríguez-Rubio et al., 2012). Mao et al. (2013) increased the lytic activity of Ply187 in milk through the development of a fusion protein CBD of Lysk (KSH3b) and the EAD of Ply187. Using high-throughput synthesis and screening of chimeric endolysins, a random domain-swapping approach coupled with domain shuffling has been developed. Yang et al. (2015a) developed an in vitro screening platform using a two-vector E. coli expression system. Using the in vitro screening platform, they reported ClyR, a new chimeric endolysin that showed a wide, evident lysis zone in the Streptococcus dysgalactiae lawn. In pasteurized milk, ClyR demonstrated a wider host range and lytic activity against Streptococcus spp. Lee et al. (2021) developed a library of chimeric endolysins by rearranging the domains of 12 native staphylococcal endolysins. ClyC, the most efficient chimeric endolysin, maintained its significant antibacterial action in milk, and animal blood, as well as protecting against MRSA-induced bacterial infection in an in vivo model of mouse infection.

Since the cell lysis of Gram-negative bacteria through natural endolysin therapy is limited, the basic objective of endolysin engineering for targeting Gram-negative bacteria is to enter the outer membrane (Oliveira et al., 2012). Endolysins targeting Gram-negative bacteria have previously been proven and showed synergistic activities through physical hydrostatic pressure (Nakimbugwe et al., 2006). Gram-negative endolysin was also reported which showed synergistic activities in combination with citric acid, chloroform, EDTA, Triton X-100, trichloroethane (CHCL3) (Briers et al., 2014; Yang et al., 2015b; Guo et al., 2017).