Conventional and Innovative Methods for Reducing the Incidence of Listeria monocytogenes in Milk and Dairy Products

Abstract

Listeriosis, the disease caused by the bacterium L. monocytogenes, can take invasive forms, with severe complications such as septicemia or meningitis, mainly affecting vulnerable people, such as pregnant women, the elderly, and immunocompromised people. The main transmission is through the consumption of contaminated food, and unpasteurized dairy products are common sources of infection. Due to the high mortality and the difficulty in eliminating the bacterium from the production environment, rigorous hygiene and control measures are essential to prevent the spread of Listeria in the food chain, and research on biofilm formation and bacterial resistance is vital to improve food safety. Dairy products, raw milk, and soft cheeses are among the most vulnerable to contamination with L. monocytogenes, especially due to pH values and low-temperature storage conditions. This paper presents a synthesis of the specialized literature on methods to reduce the incidence of L. monocytogenes in milk and dairy products. Conventional strategies, such as pasteurization and the use of chemical disinfectants, are effective but can affect food quality. Specialists have turned their attention to innovative and safer approaches, such as biocontrol and the use of nonthermal methods, such as pulsed electric fields, irradiation, and nanotechnology. Barrier technology, which combines several methods, has demonstrated superior efficiency in combating the bacterium without compromising product quality. Additionally, lactic acid bacteria (LAB) and bacteriocins are examples of biopreservation techniques that provide a future option while preserving food safety. Natural preservatives, especially those derived from plants and fruits, are promising alternatives to synthetic compounds. Future solutions should focus on developing commercial formulations that optimize these properties and meet consumer demands for healthy, environmentally friendly, and clean-label products.

1. Introduction

As foodborne illnesses remain a global problem, rigorous monitoring of the food chain and the application of technologies to ensure both the safety and quality of products are required. According to data reported by the European Food Safety Authority (EFSA) and the Centers for Disease Control and Prevention (CDC), millions of people fall ill annually due to food contamination, which highlights the need for more effective processing methods [1,2]. Developing innovative strategies to control L. monocytogenes is currently essential for the dairy industry. Despite all the advances in pathogen control, foodborne illnesses continue to represent a major global problem. Although conventional cleaning and sanitation methods are effective, they can affect the sensorial and nutritional properties of dairy products. Thus, ongoing research is needed to develop new methods that balance antimicrobial effectiveness with maintaining food quality [3,4,5,6].

Dairy products with nutritional and health claims, such as those with added probiotics, prebiotics, synbiotics, and postbiotics, along with reduced sugar, low fat, and whey protein variants, play an important role in today’s dairy industry. Consumers are increasingly concerned about health and food safety, seeking nutritious products that are free from pathogenic microorganisms. In this context, additives and new technologies that limit or eliminate pathogens are of increasing importance [7,8,9,10].

Listeriosis, an infection caused by L. monocytogenes, is frequently associated with the consumption of dairy products [11]. It is crucial to reduce the pathogen’s entry into dairy products since listeriosis, one of the most serious illnesses brought on by LM, has high rates of morbidity (>90%) and mortality (18–30%) [12]. For example, in the USA, an outbreak of listeriosis associated with queso fresco cheese was reported in the states of New York, Maryland, and Virginia, causing 12 infections and one death (CDC, 2021). In the EU, the incidence of L. monocytogenes in dairy products was reported at 0.44% between 2017 and 2020 (EFSA, 2021) [2,12].

The regulations imposed for the handling of foods contaminated with L. monocytogenes are essential, given the difficulty of eradicating this bacterium. The regulations in force cover personal hygiene, surface cleanliness, and processing technology. To date, several methods, listericidal and listeriostatic, have been developed to reduce and prevent the incidence of L. monocytogenes in milk and dairy products [13]. However, many of these methods are considered too harsh, affecting the quality and nutritional attributes of the food. In recent years, consumers have shown increased interest in minimally processed products, without additives but stable on the shelf, with improved nutritional and sensory value. To meet these demands, new strategies for the control of L. monocytogenes have been developed based on nonthermal, natural, or biocontrol methods [14,15,16].

Thermal methods such as pasteurization, sterilization, refrigeration, or freezing, although effective in eliminating pathogenic microorganisms, can affect food quality by causing undesirable chemical reactions with the formation of undesirable compounds [17]. For example, pasteurization is an effective method for eliminating pathogens from food, especially from milk, and is widely used to reduce risks to consumer health [18]. However, exposure to high temperatures can damage the chemical composition and sensory quality of food products, and there is a risk of recontamination during post-pasteurization processing. Thus, a combination of methods is needed to prevent contamination of milk and dairy products [19].

Other unconventional physical methods, such as UV irradiation, ozone, atmospheric cold plasma, high-pressure processing, pulsed electric field, and ultrasound treatments, have proven effective in extending the shelf life of foods and reducing the risk of contamination. These methods, combined with antimicrobial agents, prevent the growth of surviving bacterial cells. However, the application of physical methods can negatively affect the quality of products, especially sensory properties, such as appearance, color, or texture [19]. Chemical treatments, such as the use of caustic substances, acids, and chlorine-based disinfectants or quaternary ammonium chemical agents, although effective, can have negative effects on human health, increasing the risk of cancer. For this reason, researchers have focused on developing safer, natural methods [9]. On the other hand, the effectiveness of these measures is sometimes compromised in areas that are difficult to access [13,20]. Studies have shown that a combination of plant-derived antimicrobial compounds, along with heat treatments, can effectively control bacteria in various food products. In addition, another study showed that the combined application of pulsed electric fields, moderate heat, and natural essential oils was effective in inactivating L. monocytogenes [21]. Figure 1 presents a summary of methods to reduce the incidence of L. monocytogenes species in milk and dairy products.

Biocontrol methods represent an alternative solution to inhibit the growth of L. monocytogenes [13]. Biopreservation using LAB can ensure food safety without negatively affecting consumer health or product quality and can extend the shelf life of food products. This method is particularly promising for minimally processed products, which depend on refrigeration to prevent the growth of L. monocytogenes [22]. For example, lysozyme, a natural antimicrobial present in mammalian milk, can be used as a biopreservative in cheeses. Lactoferrin, another natural antimicrobial agent, can inhibit the growth of L. monocytogenes and other pathogens by binding the iron they need for growth [8,23].

Improving food quality and safety is essential for human well-being, with a long history dating back to prehistoric times when humans began to domesticate plants and animals and preserve food through various physical methods. Food additives and modern technologies play a crucial role in ensuring an accessible, tasty, and safe supply. These additives allow for the extension of food shelf life, reduction of losses, and development of new food formulations, meeting increasingly stringent market requirements. Despite the controversy surrounding their safety, additives are heavily regulated globally, and many have been banned over the years [24].

In the food industry, color additives and preservatives are among the most important factors in improving the appearance and preservation of products. They can be divided into antimicrobials, antioxidants, and anti-aging agents, each of which has the role of preventing or delaying food degradation. Research aims to identify new solutions that combine these properties with bioactive functions, such as antioxidant or antimicrobial activity. Although the high concentrations required for these benefits may exceed the permissible limits, the development of new chemical molecules or the modification of known natural ones may represent a solution for improving food additives [25]. Polyphenols, alkaloids, and other plant components are considered promising sources of natural preservatives, have antiseptic effects, and are generally well-accepted by consumers [26].

In many cases, a single method is not sufficient to effectively control L. monocytogenes [27]. A combination of techniques, known as “barrier technology”, has proven more effective. Combining conventional and innovative approaches can generate synergistic effects, targeting multiple critical aspects of the microorganism’s metabolism. Several studies have shown the success of these methods in controlling L. monocytogenes in foods and food production environments [22,28].

This paper deals with the review of the literature on methods used to reduce L. monocytogenes in milk and dairy products.

2. Thermal Methods

Thermal methods used in the dairy industry are divided into conventional and unconventional methods.

Table 1. Conventional thermal methods applied in the dairy industry.

Method	Description	Product	Reference
Thermalization	Preheating procedure in which milk is heated at low temperatures for a brief time prior to further processing: 57–68 °C /10–20 s	milk	[33]
	Heat treatment of milk at 57–68 °C /15 s	cheese	[34]
	Heat treatment of sheep milk at 62 °C /20 s or 68 °C/20 s	cheese	[34]
Pasteurization	Pasteurization LTLT (low temperature, long time)—the process of heating every particle of milk to at least 63 °C/30 min. Pasteurization HTST (high temperature, short time)—the process of heating every particle of milk to at least 72 °C/15 s Vacuum pasteurization—90.5–96.1 °C under vacuum pressure 62.8 °C/30 min. or 71.7 °C/15 s	milk	[35,36,37,38]
	65.6 °C/30 min. or 74.4 °C/15 s	cream	[38]
	68.3 °C/30 min. or 79.4 °C/25 s	ice cream mix	[38]
	Milk before fermentation: 90–95 °C/ 5–10 min. Milk before fermentation: 85–95 °C/15 min.	yogurt	[36,39]
	Heat treatment of milk at 63 °C /30 min.	soft cheese	[36]
	Heat treatment of milk at 72 °C, no hold	semi-hard cheese	[36]
	Heat treatment of milk at 64–68 °C/10 s	hard cheese	[36]
	Heat treatment of cream at 65 °C /10 min.	butter	[36]
Sterilization	Conventional method: Packaging is undertaken before heat treatment. The processing is usually carried out at 105–110 °C /30–45 min. UHT or aseptic method: Packaging is undertaken after heat treatment. For the ultra-high temperature and short time (UHTST) and very high temperature and short time (VHTST), the processing is at 135–150 °C/1–20 s	milk	[35,36]
Refrigeration	Artificial cooling of foods to temperatures below their freezing point Basic requirement for the processing and storage of milk and milk products is low temperature in the cold storage depending on the type of product to be stored. For example, milk is stored at 3–4 °C	milk	[40,41]
	2–8 °C	cheese	[42]
Freezing	Artificial cooling of food to temperatures at which a sufficient amount of water solidifies, stopping the activity of microorganisms Ice cream is stored at −30 °C.	ice cream	[43]
	Freeze concentration	skimmed milk skimmed powder milk fermented dairy beverages probiotic fresh cheese whey protein ice cream	[44]

presents conventional thermal methods applied in the dairy industry. Among the conventional thermal processing methods, pasteurization and sterilization are frequently used to control L. monocytogenes in food products [13,29]. Pasteurization, performed at temperatures between 60 and 80 °C, destroys microorganisms and inactivates enzymes, while sterilization, at temperatures above 100 °C, destroys bacterial spores. The D value measures the time required to eliminate 90% of microorganisms, and a higher D value indicates increased thermal resistance. For example, in milk, the D value of L. monocytogenes varies between 1683.7 s at 52.2 °C and 0.7 s at 74.4 °C [22]. Post-packaging pasteurization is gaining popularity to reduce the risk of contamination after processing. This involves heating already packaged foods with steam or hot water, but not all foods withstand high temperatures without losing their sensorial qualities. Among the unconventional technologies, microwaves, radio frequencies (RF), ohmic heating, and direct steam injection have been introduced to maintain the sensory and nutritional characteristics of food products. Microwaves and radio frequencies use non-ionizing radiation from the electromagnetic spectrum, between 30 and 300 MHz for RF and 300 MHz and 300 GHz for microwave. These technologies use uniform, volumetric, and non-contact heating of food products, which is effective against microorganisms [30]. Studies have shown that microwaves at 915 MHz can destroy L. monocytogenes and other bacteria while maintaining product quality by denaturing cell proteins and disrupting the bacterial membrane [31]. Also, Awuah et al. (2005) [32] evaluated the effectiveness of RF for inactivating L. monocytogenes in milk, obtaining a reduction of up to 5 log at 1200 W, 65 °C, and 55.5 s.

Ohmic heating, or Joule heating, treats foods by passing an electric current through them. The efficiency of this process depends on factors such as the composition of the food, its electrical conductivity, and temperature. In liquid foods, conductivity increases with temperature but decreases with increasing viscosity. Tian et al. (2018) [45] studied microbial inactivation by ohmic heating [45], and Pereira et al. (2020) [46] showed that the treatment of whey beverages by this method is more effective in reducing L. monocytogenes and has less impact on sensory and nutritional qualities [46] compared to conventional methods [22]. Ohmic heating (OH) is an interesting method of food processing because it produces heat when an electric current is passed directly through the food. The electrical conductivity of the material, the size of the product to be treated, the heat capacity and heating rate of the system, the electrode material, and the combination of applied current and voltage are all important factors that influence the rate and efficiency of ohmic heating. All of these parameters work together to create a well-performing ohmic heating system [47].

Direct steam injection, a UHT technology, allows for rapid and precise heating of liquids by directly injecting steam into the product. This is considered an effective method for preserving the nutritional qualities of milk, although it has disadvantages such as high cost and operational complexity. The water used for steaming must comply with strict hygiene standards to prevent contamination of the finished product [48].

RF technology has seen a significant increase in industrial use for food processing. Initially used for drying, disinfection, and defrosting, RF has been applied in recent years in pasteurization and sterilization processes, offering promising results for a variety of food products [49]. A better understanding of the influence of fat content and food structure on RF thermal inactivation will contribute to the development of more effective treatments [50]. To date, the use of RF has been studied for the treatment of various dairy products, such as milk [33,35,36,40,41,51,52], yogurt [36,39,53], goat milk [54], skimmed or whole milk powder [43,44,55,56,57,58], and infant formula [20,59,60,61].

3. Nonthermal Methods

Thermal sterilization is widely used due to its efficiency, low cost, and universal applicability, but it can also affect the sensory and nutritional quality of food. For this reason, nonthermal sterilization technologies have been developed, such as chemical sterilization, irradiation, high hydrostatic pressure, and high-intensity ultrasound. These nonthermal methods offer advantages in preserving food quality but come with challenges, such as high equipment costs and health risks.

3.1. High-Pressure Processing (HPP)

High-pressure processing (HPP) is a nonthermal method that uses high pressures above 100 MPa without using heat to inactivate microorganisms, thus extending the shelf life of food products. The shelf life can vary from a few days to a few weeks, depending on the type of food product [62]. The process causes changes in the structure of the cell membrane, protein structure, cell morphology, and genetic mechanisms, leading to the inactivation of microorganisms. Its efficiency varies depending on the type of microorganism and the composition of the food, which is why optimizing the parameters (pressure, time, temperature) is essential for food safety. For example, this technique is more effective in liquid foods than in solid foods. However, HPP does not always guarantee the complete destruction of microorganisms, and sublethal cells can survive and recover later. Multiple studies have demonstrated the effectiveness of HPP in inactivating L. monocytogenes at pressures of 450–600 MPa for 3–15 min, depending on the food and bacterial strain. However, a treatment of 375 MPa for 15 min was not sufficient for pasteurization of milk and poultry meat [63].

High-pressure processing (HPP) has minimal effects on food components, preserving natural flavors and a high content of nutrients such as vitamins and antioxidants [63]. Unlike thermal pasteurization, HPP does not affect covalent bonds, which allows the preservation of the sensory and nutritional properties of foods. Treating milk with HPP leads to the breaking of ionic and hydrophobic bonds in proteins without affecting bioactive proteins or other essential nutritional components such as vitamins and amino acids. In addition, HPP causes beneficial changes such as denaturation and aggregation of proteins, influencing the yield of dairy products derived from the treated milk [64]. Recent studies have demonstrated the success of applying HPP in extending the shelf life of products such as cheddar and gorgonzola cheese [65,66].

Food composition, type and age of microorganisms, applied pressure, and duration of treatment play a crucial role in the efficiency of microbial inactivation. HPP is effective against vegetative pathogens but has limitations in inactivating spores, which can be overcome by combining HPP with other thermal or nonthermal methods. This synergistic approach preserves the nutritional quality of foods and reduces processing intensity. Although HPP equipment is expensive, technological advances and the increase in the number of HPP units have contributed to the commercialization of this technology, especially in developed countries. Studies have also shown that other nonthermal pasteurization methods, such as ultraviolet radiation, pulsed electric field (PEF), and ultrasound, are less effective than HPP. For example, PEF and ultrasound require moderate temperatures to increase the efficiency of microbial inactivation, and ultraviolet radiation has low penetration in opaque fluids. Membrane filtration also involves high maintenance costs and is less efficient in treating large volumes of liquids [67].

3.2. Ultrasound Method

Ultrasound is another efficient and environmentally friendly nonthermal technology, which is essential in the food industry to meet consumer demands for safe and healthy food and an innovative method for food preservation and safety. Ultrasound shows great potential in the decontamination of food pathogens, such as L. monocytogenes, Salmonella, and Staphylococcus spp. However, to optimize this technology, parameters such as frequency, intensity, and treatment time must be adjusted according to each type of food and microorganism involved [68].

The use of high-power ultrasound (20–100 kHz) causes the destruction of microorganisms by cavitation, a process in which gas bubbles formed in liquids collapse, generating shock waves that destroy the cell walls of microorganisms and modify their DNA. A study demonstrated the effectiveness of ultrasound in reducing L. monocytogenes biofilm on stainless steel surfaces, with a decrease of 3.8 log cfu/mL. Also, combining ultrasound with ozonation led to a reduction of 7.31 log cfu/mL, with no recoverable cells after treatment [10].

The use of ultrasound in dairy products has increased due to its effects on (a) the thermal stability of whey proteins, (b) the gelation of caseins, (c) the viscosity of the dairy product, (d) the homogenization of milk, and (e) the reduction of the freezing time of ice cream [69].

Recent studies suggest that ultrasound, combined with other technologies within the framework of the “multiple hurdle technology”, improves the efficiency of the microbial inactivation process compared to the use of a single technology. This requires further research on the mechanisms of inactivation and the synergistic effects of different technologies. Thus, ultrasound becomes a promising method of nonthermal sterilization, capable of improving food safety while preserving the quality of the finished product [70].

3.3. Pulsed Electric Field (PEF)

Pulsed electric field (PEF) is another nonthermal method used to control L. monocytogenes. This technique inactivates microorganisms by applying high-voltage electric field pulses (>18 kV/cm) for a short period. PEF disrupts the bacterial cell membrane, resulting in the destruction of the microorganism. Factors that influence the effectiveness of this method include electric field strength, temperature, and pH [71,72]. One study showed that PEF is more effective at lower pH and higher electric field strengths [73]. For example, at pH 3.5, a treatment of 28 kV/cm for 400 s resulted in a 6 log10 reduction in L. monocytogenes cells. PEF technology has been used in various food products, including milk, juices, and soups [22].

3.4. Ionizing Irradiation

Ionizing irradiation is another method used to eliminate L. monocytogenes from the food chain, in which products are exposed to ionizing radiation, such as gamma rays, high-energy electrons, or X-rays. Irradiation involves exposing food products to gamma radiation to destroy L. monocytogenes and other bacteria that can cause food poisoning or food spoilage. Irradiation has been shown to be more effective when applied to frozen foods, as it allows a higher dose to be delivered before undesirable sensory changes occur. In another study, electron beam irradiation reduced the bacterial load in soft cheeses, but the affected cells recovered during storage. Combining irradiation with other methods, such as the application of nisin, has been shown to be more effective in eliminating L. monocytogenes [74,75].

3.5. Ultraviolet (UV) Radiation

Ultraviolet (UV) radiation is used to eliminate microbial contamination from surfaces, air, and water and is approved for reducing microbial loads in foods and juices. UV-C light (254 nm) alters the DNA of microorganisms, preventing their reproduction. The effectiveness of UV varies depending on the surface of the food. For example, studies have shown that UV-C is also effective for decontaminating surfaces in the food industry, including stainless steel [76].

3.6. Ozone Using Method

The application of ozone is another effective method, an environmentally friendly solution classified as generally recognized as safe (GRAS), being appreciated for its ability to reduce microorganisms due to its strong oxidative properties. The use of ozone in the dairy industry is considered a clean technique, capable of eliminating microorganisms and limiting enzymatic activity without leaving chemical residues and increasing efficiency in controlling microbial loads. Ozone can also be used to sanitize equipment, helping to prevent contamination and ensure food safety. Studies have shown that the application of ozone in the food industry, including in the cleaning and sterilization processes of equipment, can significantly reduce the microbial load without affecting the quality of the products [14].

In a recent study, the use of gaseous ozone at 50 ppm on food-associated strains of L. monocytogenes resulted in a significant reduction in bacterial load, with a decrease of more than 3 log10 cfu/mL after 10 min. Also, after 6 h of treatment, a complete inactivation of planktonic cells and a considerable reduction in biofilm biomass were observed [77]. Thus, gaseous ozone is a promising method for controlling L. monocytogenes contamination on food contact surfaces and in finished products [78].

The effectiveness of ozone can vary depending on factors such as temperature, pH, humidity, and the presence of organic matter. Also, the inactivation of microorganisms by ozonation is a complex process that depends on the cellular structures and composition of the microorganisms. In the case of L. monocytogenes, a food pathogen with a high potential to contaminate dairy products, ozone has been shown to be effective in reducing microbial loads. In a study conducted in a cheese factory, ozonation significantly reduced the presence of L. monocytogenes in the samples tested. Ozone was used in both gaseous and aqueous forms, demonstrating its effectiveness against a wide range of pathogenic microorganisms [79].

A study by Muthuchamy (2011) [35] showed the complete elimination of L. monocytogenes in raw and processed milk after 15 min of ozone treatment. Cavalcante et al. (2013) [80] also observed a reduction in bacteria, including Listeria and Enterobacteriaceae, in ozone-treated milk. Morandi et al. (2020) [81] applied ozone gas to various cheeses and observed a significant reduction in L. monocytogenes.

However, some studies suggest that although ozone reduces microbial loads, its effectiveness may be limited in certain cases, requiring combination with other methods. As research continues, it is anticipated that ozonation will become a standard method of food sterilization and preservation due to its efficiency and low environmental impact [14].

3.7. Sonodynamic Technology (SDT)

Sonodynamic technology (SDT), originally developed in the medical field for the treatment of cancer, is now being explored for its applicability in food safety. SDT uses ultrasound to activate sonosensitizers, generating reactive oxygen species (ROS) that destroy the cellular structures of microorganisms. Sonosensitizers can be used for food sterilization, being effective in combating bacteria and viruses without affecting the quality of food products [6].

3.8. Intense Pulsed Light (IPL)

Intense Pulsed Light (IPL) sterilization technology, a nonthermal method, uses a gas lamp (e.g., Xenon or Krypton) to generate light with wavelengths similar to sunlight. IPL, also known as high-intensity pulsed ultraviolet light, effectively reduces microbial contamination of food. Studies show that IPL treatment has resulted in significant reductions in pathogens such as Salmonella, Campylobacter, Escherichia coli, and Listeria. The efficiency varies depending on factors such as treatment conditions and product characteristics, with minimal impact on the quality of the treated food products [82].

All these modern nonthermal methods not only meet the increased requirements for safety and quality but also contribute to environmental protection by reducing the use of chemicals and energy consumption. Therefore, nonthermal technologies offer efficient and innovative solutions for the control of L. monocytogenes contamination in the food industry. The combined use of these methods can provide more effective control, reducing the risks of food poisoning and extending the shelf life of products. These approaches contribute to increasing food safety while preserving the quality and freshness of food products [83].

4. Biocontrol Methods

Biological methods inhibit the growth of pathogenic bacteria, including L. monocytogenes, and contribute to maintaining food quality. Microbial sources include bacteriophages, LAB, and probiotics and their metabolites, and probiotics are considered safe for consumption, according to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) [84]. In addition, they do not negatively affect food safety. Probiotics, as part of natural preservation methods, are preferable to chemical preservatives (e.g., sorbates, benzoates, nitrites), which have been associated with risks to human health, such as genotoxic effects or cancer risk [85].

4.1. Lactic Acid Bacteria

Biocontrol, by using LAB to prevent the growth of pathogenic microorganisms, is an increasingly explored strategy in the dairy industry. LAB are effective in inhibiting Gram-positive bacteria, and in the case of artisanal cheeses made with raw milk, they have been used to control foodborne pathogens. LAB produce bioactive metabolites, such as organic acids, antimicrobial peptides, and enzymes, which contribute to the inhibition of pathogens and the extension of the shelf life of products [86].

LAB, a large group of non-sporulating Gram-positive bacteria, such as Lactococcus, Lactobacillus, Enterococcus, Streptococcus, and Leuconostoc species, are recognized for their ability to produce lactic acid, antimicrobial peptides, and bacteriocins, having a long history of use in the production of fermented foods and being proven to inhibit or destroy food pathogens, such as L. monocytogenes and Staphylococcus aureus [87,88]. Among them, lactobacilli and lactococci are among the most important LAB species, with beneficial effects on human health. They are generally considered safe (GRAS) and can be used directly in food products. LAB metabolites contribute to the inhibition of the growth of L. monocytogenes through bactericidal and bacteriostatic mechanisms. Advanced metagenomics, proteomics, and metabolomics studies are needed for a better characterization of probiotic bacteria and their role in maintaining intestinal health.

Homofermentative LAB produce lactic acid as their main metabolite, reducing pH and inhibiting the growth of L. monocytogenes. Heterofermentative bacteria produce, in addition to lactic acid, ethanol and carbon dioxide, compounds that contribute to the inhibition of bacterial growth by disrupting the cell membrane and denaturing proteins necessary for bacterial survival [89].

Lactic acid and acetic acid are the most important antimicrobial acids, reducing the intracellular pH of pathogenic bacteria and causing the death of microbial cells. Although L. monocytogenes possesses acid tolerance genes, lactic acid at high concentrations can cause their inactivation. For example, a concentration of 0.5% lactic acid reduced the population of L. monocytogenes by 6 log₁₀ cfu/g in just two hours. LAB also produce antimicrobial compounds such as hydrogen peroxide, which enhances antimicrobial effects by activating the lactoperoxidase system. This system generates hypothiocyanite anions that destroy bacterial cellular components. Also, diacetyl, produced by LAB, interferes with the utilization of arginine by bacteria, leading to their death. The consumption of oxygen by LAB creates an anaerobic environment unfavorable for aerobic bacteria [90].

In the case of artisanal cheeses, the use of LAB has been well documented, contributing not only to the control of pathogens but also to the improvement of the nutritional and sensory profile of the products. LAB can degrade harmful compounds such as biogenic amines and cholesterol, contributing to the increase in the concentrations of beneficial substances such as antihypertensive peptides, GABA (aminobutyric acid), and short-chain fatty acids. The latter is essential for metabolic health, and the ability of LAB to produce such compounds makes them a key element in the food industry [86].

Regarding bacterial competition, LAB grow faster than L. monocytogenes and compete effectively for nutrients, negatively affecting the growth of the pathogenic microorganism. Studies show that LAB inhibit L. monocytogenes in various food matrices, especially in meat and ready-to-eat (RTE) dairy products. For example, Pisano et al. (2022) [91] isolated several strains of Lactococcus and Lactobacillus from dairy products from Sardinia, observing their anti-listerial activity in fresh cheeses. Three strains significantly reduced L. monocytogenes by 4 log cfu/g, and other strains had a bacteriostatic effect. The study carried out highlighted the importance of selecting and specifically testing LAB strains in food matrices since their in vitro efficiency may differ from that in real products.

Panebianco et al. (2022) [92] reported that native strains of LAB from Calabria effectively inhibited L. monocytogenes in cheeses, and strains such as Lactobacillus sakei and Lactobacillus plantarum reduced the pathogenic load by 0.5–1.0 log cfu/g. The importance of these studies lies in the fact that the selected strains do not affect the fermentation capacity of the initial cultures, improving food safety without compromising their quality.

In another study, Lactococcus lactis, combined with lactic acid or sodium lactate, was found to be effective against L. monocytogenes on gorgonzola cheese at 4 °C. The combination reduced the pathogen load below the detection limit after 60 days of ripening. Carnobacterium divergens was also tested, and greater antimicrobial activity was observed as the pH of the cheese increased during ripening, indicating the potential for the sequential use of LAB strains at different stages of production [81].

The use of Lactococcus spp. in dairy products for biopreservation has increased significantly. For example, in Moroccan fermented milk, the addition of L. lactis completely inhibited L. monocytogenes within 24 h of storage at 7 °C, even in the case of high initial contamination. L. plantarum, a plantaricin producer, has also been shown to be effective in inhibiting L. monocytogenes in cheeses, especially in combination with nisin producers, reducing the pathogen load below detectable levels within 4 weeks [93].

Other studies have investigated the use of LAB in raw milk cheeses, which are appreciated worldwide. Selected strains, such as Lactobacillus brevis and Enterococcus faecalis, reduced L. monocytogenes by 4 log cfu within a few weeks and demonstrated efficacy during cold storage of soft cheeses. Pathogen reduction is also influenced by the genus of LAB and the type of food, which highlights the need to test strains in each specific food category [94].

Studies have shown that L. brevis, L. plantarum, and E. faecalis have bacteriostatic effects on L. monocytogenes in soft cheese and bactericidal effects in semi-hard cheese, with promising results for periods of up to 20 days. Also, the addition of LAB and lactic acid (a safe food additive) to ripened cheeses can inhibit the growth of L. monocytogenes for long periods [95].

Another mechanism for controlling L. monocytogenes in biofilms is the use of competitive exclusion bacteria. Zhao et al. (2004) [96] demonstrated that a combination of L. lactis and Enterococcus durans significantly reduced L. monocytogenes in biofilms over a wide range of temperatures. These bacteria are able to survive and compete with L. monocytogenes in food environments, offering a practical and economical solution for preventing contamination with this pathogen [97].

4.2. Probiotics

Probiotics, including bacteria of the genus Bifidobacterium and probiotic yeasts, inhibit the growth of harmful bacteria such as L. monocytogenes and can be used to maintain the original properties of food products, including dairy products such as milk and cheese. They also improve the flavor and extend the shelf life of foods. In recent years, research has highlighted the role of probiotics in food preservation, especially in dairy and meat products. Although probiotics cannot completely replace existing preservatives, they can help reduce their use. Probiotics are a promising solution to control contamination with L. monocytogenes and other pathogenic bacteria, thus offering a safer and less invasive alternative to chemical preservatives [98,99].

Although there are numerous studies that have demonstrated the effect of probiotics against pathogenic bacteria in vivo, their effects on L. monocytogenes in vitro are not as well documented [100]. Current research has mainly focused on LAB, but there are few data available on the activity of other probiotics, such as Bifidobacterium and yeasts, on L. monocytogenes. In addition, the mechanisms of virulence inhibition and biofilm formation by probiotics are not systematically described [101,102].

Another aspect that requires attention is the effect of probiotics on the adhesion of pathogenic bacteria and on biofilm formation. Although bacteriocins produced by probiotics have shown the ability to inhibit biofilm formation, the specific mechanisms by which probiotics influence these processes remain unclear and require further investigation [98,103].

Probiotics, such as Lactiplantibacillus plantarum, Lactiplantibacillus sakei, and Lacticaseibacillus rhamnosus, have been shown to be effective in controlling the growth of L. monocytogenes in various food products. Dairy products, which are ideal substrates for these pathogenic bacteria due to their high water activity and low preservative content, are an area where the effects of probiotics have been intensively studied. Studies have shown that probiotics can inhibit the growth of harmful microorganisms in these products, thus extending their shelf life [104].

Although probiotics inhibit the colonization of L. monocytogenes, they do not always completely eliminate these bacteria. In this context, the combined use of probiotics with other antimicrobial methods (chemical, biological, or physical) may have a synergistic effect, compensating for the possible shortcomings of each method used individually. For example, the combination of probiotics with physical treatments, such as ultraviolet radiation or the use of hydrogen peroxide (H₂O₂), could enhance the antibacterial effects. However, some of these methods could inactivate probiotics, which requires careful screening of combined methods to avoid diminishing the effect of probiotics [85].

Research has highlighted the effect of probiotics on the reduction of L. monocytogenes in dairy products such as Gorgonzola and Minas cheese. The effectiveness of LAB, such as Lactobacillus rhamnosus, in controlling L. monocytogenes has been demonstrated, as they have excellent resistance to acids and bile salts, making them effective in food environments [105]. Probiotic metabolites, such as organic acids and exopolysaccharides, have shown significant antimicrobial effects against L. monocytogenes. However, the stability and efficacy of these compounds under real food processing conditions remain to be evaluated [106,107].

Prevention of L. monocytogenes contamination in the dairy industry through the use of probiotics requires studies that take into account pathogenic variations between different bacterial strains and specific food processing conditions, such as temperature and storage period. In addition, it is necessary to explore combinations of probiotics that provide increased efficacy against a broader spectrum of pathogens. Current studies have shown that the use of probiotics to control L. monocytogenes in dairy products can lead to safer and healthier foods with improved organoleptic characteristics. However, the use of probiotics needs to be studied in depth to fully understand the effects on the sensory and nutritional properties of foods [108].

The application of LAB in food packaging represents an innovative strategy to prevent the growth of pathogenic germs. Despite the benefits, some studies have identified adverse effects related to the use of probiotics, such as metabolic disorders and the production of biogenic amines.

4.3. Postbiotics

Thus, the use of postbiotics, i.e., metabolites of LAB, has been proposed as an alternative. Postbiotics are compounds produced by probiotic bacteria, such as peptides, vitamins, and fatty acids, with health benefits, including anti-inflammatory, antioxidant, antihypertensive, and anti-obesity effects [109].

Encapsulation of postbiotics in films increased their solubility in water and the opacity of the films without altering their mechanical properties. The incorporation of postbiotics or bacteriocin-rich extract demonstrated significant antibacterial activity [110]. Divsalar et al. (2018) [111] used cellulose paper with chitosan-ZnO nanocomposite and nisin (500–1000 μg/mL), obtaining a strong inhibition of L. monocytogenes. After 14 days of storage at 4 °C, nisin films completely inactivated L. monocytogenes on the surface of white cheese [112].

Hurdle technology is used to achieve cumulative antimicrobial effects in foods, improving food safety by reducing the intensity of treatments and preserving product quality [113]. LAB easily adapt to various processing and storage conditions, such as refrigeration, low pH, and salting, which makes them useful in this technology. Their ability to produce antimicrobial substances allows them to be an important element in food safety [88].

Bacteriocins are ribosomally produced peptides by LAB, resistant to heat and acids, with antimicrobial activity against pathogenic bacteria in food products by creating pores in the cell membrane and inhibiting DNA replication and protein synthesis [114]. Bacteriocins are effective antimicrobial compounds capable of inhibiting pathogenic bacteria, such as L. monocytogenes, through various mechanisms, such as changing the permeability of the cell membrane, inhibiting cell wall synthesis, and interfering with metabolic processes [61]. Depending on the gene sequence and amino acid composition, bacteriocins are classified into four classes: class I (lantibiotics) below 5 kDa, heat stable, with lanthionine; class II (unmodified peptides) below 10 kDa, heat stable, without lanthionine; class III (large and thermolabile peptides) proteins above 30 kDa, heat sensitive; and class IV (cyclic peptides), bacteriolysins [24].

A notable example of a heat-stable bacteriocin is nisin, a lantibiotic produced by L. lactis, approved for use as a food additive (E234), generally recognized as safe for human consumption (GRAS) by the World Health Organization and the FDA, and used in over 50 countries. It acts by interfering with bacterial cell wall biosynthesis, being effective against pathogens such as L. monocytogenes and Staphylococcus aureus, and is used in various food products such as processed cheeses [115]. Nisin forms pores in the membrane of pathogenic cells, thereby blocking cell wall synthesis and inhibiting bacterial growth. However, the use of nisin, a natural antibacterial agent, is limited in dairy products with neutral pH, such as whole milk or fresh cheese. The effectiveness of nisin decreases in environments with pH above 6, and in queso fresco, nisin did not effectively inhibit the growth of L. monocytogenes [116]. Also, the presence of divalent cations such as calcium and magnesium and the fat composition of dairy products influence the activity of nisin, reducing its potency, especially in the case of homogenized milk. This phenomenon is explained by the absorption of nisin by the homogenized fat globules, which decreases the antibacterial efficiency. Under these conditions, more studies are needed to determine the impact of factors such as pH, fat content, and divalent cations on the activity of nisin in other dairy products [9].

An important aspect of the application of probiotics and bacteriocins in the food industry is the protection of these compounds from harsh processing conditions [117]. For example, nisin is widely used in various food products such as meat, dairy, and aquatic products due to its antibacterial efficacy against Gram-positive bacteria, including L. monocytogenes. Its unique chemical structure gives it thermal stability and resistance to proteolytic digestion, making it effective in maintaining the integrity of the bacterial cell membrane. In addition, nisin combined with other methods, such as physical treatments (e.g., electric field) or chemical agents (e.g., organic acids), has been shown to broaden its antimicrobial spectrum and reduce the amount of nisin required in food.

Studies have reported that nisin can be enhanced in its antibacterial efficacy by combining it with other bacteriocins, inorganic agents, or multiple methods, such as high-intensity ultrasound or UV-A light. For example, combining nisin with these technologies provided enhanced antibacterial protection without significantly affecting product quality. However, in some cases, these combinations altered the sensory characteristics of dairy products, such as color and taste, or even texture, as was the case with some cheeses treated with encapsulated nisin over the long term. The efficacy and safety of encapsulated nisin need to be further evaluated, particularly with modern antibacterial packaging methods, which could include antibacterial agents integrated into polymers.

Although nisin has demonstrated antibacterial activity against L. monocytogenes, its use is limited due to the high cost of isolation and purification, narrow spectrum of activity, and the risk of developing nisin resistance. Another obstacle is the potential toxicity associated with high concentrations of nisin. Nisin efficacy also varies with pH, temperature, food composition, and microbiota composition of the food product, and future research should focus on optimizing these variables.

Experts recommend exploring other strategies for applying nisin, such as using nisin-producing strains in fermented foods or adding encapsulated nisin in various forms to control its gradual release and protect it from proteolytic degradation. Combining it with other antibacterial techniques may enhance efficacy while limiting adverse effects on the organoleptic characteristics of dairy products and preventing the development of nisin resistance (CDC, 2021) [9]. In addition, combinations of nisin with other natural substances, such as sesamol or carvacrol, have shown synergistic effects, increasing its antimicrobial efficacy. Recent studies have evaluated the efficacy of nisin in combination with lauric alginate and ε-polysine in cheeses. Martínez-Ramos et al. (2020) [118] demonstrated that these combinations can effectively control L. monocytogenes in fresh cheese for periods of up to 28 days of cold storage. The use of phytic acid in combination with nisin has also been investigated to increase its efficacy against Gram-negative bacteria, such as Escherichia coli [119].

Bacteriocins can be used in the food industry either by adding the producing strains to food or by extracting them in purified or semi-purified form. Studies indicate the effectiveness of bacteriocins, including nisin and pediocin, in inhibiting L. monocytogenes in meat, fish, dairy products, salads, and juices [120]. For example, the application of bacteriocins on packaging prevented the growth of L. monocytogenes for up to 12 weeks at 4 °C [22].

These liposomes reduced the number of L. monocytogenes from 4.5 log cfu/mL to an undetectable level in whole milk and skim milk after 14 days of refrigerated storage at 7 °C. The antimicrobial peptide P34 was also encapsulated in liposomes, which was able to control L. monocytogenes only in skim milk for up to 8 days. The antimicrobial activity of liposome-encapsulated nisin was also effective against L. monocytogenes in fluid milk by prolonging the lag phase at both 5 °C and 20 °C. In another study, liposomes and 1,2-dioleoyloxy-3-trimethylammonium-propane were used to encapsulate sakacin, controlling the growth of L. monocytogenes in goat milk for 5 days at 7 °C, with a reduction of approximately 5 logs compared to the control sample. These results are relevant since the recommended period for consumption of milk after opening the package is approximately 5 days [8]. In addition to bacteriocins, LAB produce antimicrobial compounds, including organic acids and reuterin, a compound produced by Lactobacillus reuteri. Reuterin, at a concentration of 8 AU/mL in milk, completely inactivated L. monocytogenes after 24 h of incubation at 37 °C [121]. Reuterin and nisin, in combination, also acted synergistically against L. monocytogenes in milk [97].

A novel bacteriocin, agilicin C7, produced by Ligilactobacillus agilis, has demonstrated inhibitory activity against L. monocytogenes. Agilicin C7, characterized as a glycoprotein stable at pH, temperature, and organic solvents, destroys L. monocytogenes by damaging the cell membrane. These characteristics suggest a potential application of this bacteriocin in the food industry, including dairy and meat products [97]. Compared to antibiotics, bacteriocins have a lower risk of inducing antimicrobial resistance due to their ability to directly disrupt the integrity of the bacterial membrane. This is essential in the context where antibiotic resistance is a major global problem, with estimates indicating 10 million deaths annually by 2050 if effective solutions are not found [24]. Bacteriocins, being proteins, are non-toxic and easily degradable in the human gastrointestinal tract. In addition, combining bacteriocins with heat treatments reduces the temperature and duration of the heat treatments, thus better preserving the nutrients and sensory properties of the food products.

The effect of bacteriocins varies depending on the dose and environment. For example, Pisano et al. (2022) [91] found that certain strains of LAB had high antilisterial activity in vitro, but this activity decreased in the presence of native LAB from milk. Environmental factors, such as pH, temperature, and food structure, influence the efficacy of bacteriocins, and microbial interactions in the complex food matrix can significantly affect antimicrobial activity.

Purified bacteriocins have been shown to be effective in reducing L. monocytogenes in certain products, such as fresh cheese, but their use is limited due to their low solubility and narrow antimicrobial spectrum. Furthermore, bacteriocins degrade over time, allowing pathogens to re-emerge. However, bacteriocin-producing LAB have greater potential for use in biopreservation cultures due to their ability to generate bacteriocins in situ, providing additional protection against pathogens.

There are limitations to the use of bacteriocins, including resistance of pathogenic bacteria and their interactions with other food components, such as proteins and fats. Therefore, recent research has focused on improving the effectiveness of bacteriocins by combining them with food nanotechnology. Also, the combined application of bacteriocins, probiotics, and other natural antibacterial compounds, such as tea polyphenols or essential oils, could be an effective future strategy to eliminate the risk of L. monocytogenes contamination. Although significant progress has been made in the use of probiotics and bacteriocins in the food industry, further research is needed to better understand the complex mechanisms by which these products can inhibit the biofilm and virulence of L. monocytogenes.

Recent research suggests that indigenous LAB-producing bacteriocins from traditional cheeses can be used as effective alternatives for food preservation. Also, improving the knowledge of traditional cheese microbiota and using advanced methodologies can lead to the development of more effective starter cultures and improved manufacturing practices. These findings contribute to the production of higher quality and safer cheeses for consumers.

Pediocin PA-1, a class II bacteriocin produced by Pediococcus spp., is effective against L. monocytogenes. Pediocin is marketed as a part of different preparations, such as Alta 2341TM, and is used in fermentation processes to prevent contamination of animal-derived foods, especially cheeses. Studies have shown that a combination of bacteriocins with other natural preservatives, such as reuterin, can be more effective in preventing the growth of pathogens than their individual use [122]. This approach has the potential to significantly extend the shelf life of food products and improve their safety.

The application of bacteriocins in food is, however, limited by their narrow spectrum of action and loss of activity in the presence of enzymes or unfavorable pH. Encapsulation of bacteriocins may provide a solution, protecting them from degradation and ensuring a controlled release into food.

Microencapsulation technology of bacteriocins allows their efficient application in food products, such as milk and cheese. For example, nisin-loaded zein microcapsules have been shown to be effective in reducing the population of L. monocytogenes in treated milk [5]. Encapsulation protects bacteriocins from interactions with food composition and ensures their slow release, maintaining antimicrobial activity.

Edible polymer films can be used to evenly distribute bacteriocins on food surfaces, preventing aggregation and disruption of lipid membranes. Edible coatings containing nisin have also been shown to be effective against pathogens, extending the shelf life of food products [24,123].

Therefore, bacteriocins represent a promising solution, especially since they are effective against antibiotic-resistant bacteria. With the identification of an increasing number of bacteriocins, there is a major interest in studying them to explore their applicability in the food industry [124,125]. Recently, a nanoencapsulation technique has been developed that improves the antimicrobial activity of bacteriocins, allowing them to be applied more effectively in the food industry [117]. Nanoencapsulation of bacteriocins is an effective technique that uses nanomaterials to improve the antimicrobial potential of these peptides. This method allows the application of bacteriocins in food matrices, where they interact effectively with pathogenic microorganisms. Although nanoencapsulated bacteriocins are already used in the food industry, further research is needed to better explore their potential [124,126,127].

In conclusion, bacteriocins are promising antimicrobial compounds for food preservation. Although nisin is the only bacteriocin approved by the FDA for use in food products, there is a growing interest in the discovery and use of other bacteriocins in the food industry. In addition, the development of encapsulation technologies and the combination of bacteriocins with other bioactive agents can contribute to the efficiency of their use in food products, providing safe and effective solutions for natural food preservation. Future studies should focus on expanding the spectrum of application of bacteriocins and optimizing encapsulation methods to ensure better stability and antimicrobial efficacy [128].

4.4. Bacteriophages

Bacteriophages, also known as phages, are viruses that infect and specifically kill bacteria. Bacteriophages were discovered in the late 19th century when British bacteriologist Ernest Hanbury Hankin observed the bactericidal activity of water from the Ganga and Jamuna rivers against Vibrio cholerae. In 1915, Frederick Twort, a British microbiologist, first described bacteriophages, and two years later, Felix d’Herelle used them to treat dysentery. Although the use of phages declined with the discovery of antibiotics, bacterial resistance to them has brought the potential of phages back into focus [129].

In recent decades, the use of phages to combat bacterial pathogens in food, an approach called “phage biocontrol”, has become increasingly common. For example, ListShield™ was the first commercial phage product approved by the Food and Drug Administration (FDA) in 2006, followed by Listex™ P100, to combat L. monocytogenes in ready-to-eat foods. Since then, other phage preparations have been approved by regulatory agencies in the US and other countries, including Canada and Israel.

Phages have been shown to be effective in controlling L. monocytogenes, significantly reducing its presence in foods, such as vegetables and fruits, as well as in cheese and smoked fish. Phages are also used to decontaminate meat and cheese during the ripening process. For example, studies have shown that ListShield™ can reduce L. monocytogenes contamination by 2.2 log units on prepackaged frozen foods, making it effective in a variety of applications. Other products, such as Listex™, have shown similar results in reducing bacterial contamination [130]. Phage biocontrol is considered an effective, environmentally friendly, and natural method of controlling pathogenic bacteria in food. Most commercially available phage products consist of natural lytic phages, which are not genetically modified and are considered safe for consumption. Many of these products are also certified Kosher, Halal, and organic, making them attractive to a wide range of consumers.

The low cost of phage application in the food industry makes them competitive compared to other food safety methods such as irradiation or high-pressure pasteurization. However, phage biocontrol has some limitations, such as a more modest reduction of bacterial load in the range of 1–3 log units compared to chemical disinfectants. Also, the specificity of phages makes it necessary to use several products to combat different pathogens in case of multiple food contamination. However, this specificity is an advantage, as it targets only pathogenic bacteria without affecting the beneficial microflora or food quality.

Phages have two types of life cycles: lytic and lysogenic [131]. Lytic phages are preferred for biocontrol because they destroy bacterial cells, while lysogenic phages integrate into the DNA of the host bacterium and do not cause its death. Phages do not pose a risk to humans due to their specificity for host bacteria.

Studies have demonstrated the effectiveness of phages in combating Listeria monocytogenes biofilms formed on stainless steel surfaces, which are commonly found in food processing environments. For example, ListShield™ successfully reduced L. monocytogenes biofilm in 100% of cases, and Listex™ P100 had a similar effectiveness against biofilm on stainless steel.

Phages are widely used in countries such as the United States and Canada, but their use in the European Union is still limited. Although the EFSA (European Food Safety Authority) has given a positive opinion of Listex™ P100, the product has not been approved for use in the food industry in all EU member states. This highlights the need for further research on the safety and efficacy of phages, as well as monitoring the emergence of phage-resistant strains [78].

An alternative method to using phages is to apply their enzymes, called endolysins. These enzymes destroy the bacterial cell wall and can be used to control pathogenic bacteria without involving phages entirely. Studies have shown that the endolysin PlyP100 is effective in combating L. monocytogenes in food products such as queso fresco, providing a significant reduction in bacterial load.

Combining phages with other antimicrobial methods, such as probiotics, bacteriocins, or plant extracts, can have a synergistic effect against L. monocytogenes. For example, nisin and propolis have been reported to have a synergistic effect in reducing bacteria in ice cream without affecting product quality. Other combinations, such as LAB and grapefruit seed extract, have been effective in reducing bacterial growth in fresh soft cheese [132].

Bacteriophages are considered a safe and viable alternative to traditional preservation methods, with the ability to eliminate or reduce foodborne pathogens. They can be used pre- and post-harvest, and they are being applied to various food products. In addition, they can be used to disinfect equipment and contact surfaces in the food industry, thus preventing bacterial contamination [133].

Food manufacturers have begun to integrate the use of bacteriophages into their pathogen control strategies as part of a multi-faceted approach to increasing food safety. This is due to the advantages that bacteriophages offer, such as the possibility of being applied at different stages of the production process, either by spraying or immersion. The use of natural lytic phages also allows for the selective elimination of pathogens without affecting the natural microflora of the food or its organoleptic qualities. However, some research highlights the difficulties in producing reliable commercial phage products for food applications, and research in this direction is ongoing [17].

The use of phages as biocontrol agents offers a promising solution, helping to reduce the use of chemical preservatives, which are becoming increasingly unavailable due to strict regulations. Phages offer a natural and effective method of combating pathogens, from the production farm to the final consumer. In addition to food safety applications, phages are also used in other industries for decontamination and bacterial control. However, for their widespread use, industrial-scale production processes must be safe and efficient. Studies show that under real-world conditions in food systems, phage activity can be reduced compared to ideal laboratory conditions. Therefore, sustained effort is needed to optimize their use under real-world food processing conditions.

Another important factor for the widespread adoption of phages is educating consumers and industry operators about their benefits and safety. Although phages are already being used commercially in some countries, consumer reluctance to use “viruses” in food may delay the adoption of this technology. However, as information about the effectiveness and safety of phages becomes more widely available, their use is expected to increase [123].

Table 2. Overview of treatment methods for controlling L. monocytogenes in dairy products: conditions, efficacy, and food matrices.

Treatment Method	Treatment Conditions	Inactivation Efficiency	Food Matrix	Reference
Use of LAB	General application in food; produces lactic acid, antimicrobial peptides, diacetyl, etc.	Inhibits L. monocytogenes and other pathogens	Dairy products (e.g., cheeses) and fermented foods	[86,87,88]
0.5% lactic acid	Applied for 2 h	6 log10 cfu/g reduction of L. monocytogenes	Not specified	[90]
LAB (Lactococcus, Lactobacillus) strains	Isolated from Sardinian dairy; used in fresh cheeses	4 log10 cfu/g reduction (bactericidal); others are bacteriostatic	Fresh cheeses	[91]
L. sakei, L. plantarum	Native strains from Calabria; applied in cheeses	0.5–1.0 log10 cfu/g reduction	Cheeses	[92]
L. lactis + lactic acid/sodium lactate	Gorgonzola cheese, stored at 4 °C for 60 days	Pathogen load reduced below detection limit	Gorgonzola cheese	[81]
L. lactis	Added to Moroccan fermented milk, stored at 7 °C	Complete inhibition of L. monocytogenes in 24 h	Fermented milk	[93]
L. plantarum + nisin producers	Applied in cheese; monitored for 4 weeks	Pathogen load reduced below detection level	Cheese	[93]
L. brevis, E. faecalis	Used in soft cheeses; monitored during cold storage	4 log10 cfu reduction in few weeks	Soft cheeses	[94]
L. brevis, L. plantarum, E. faecalis	Soft vs. semi-hard cheeses, over 20 days	Bacteriostatic (soft); bactericidal (semi-hard)	Soft and semi-hard cheeses	[95]
LAB + lactic acid	Applied to ripened cheeses	Long-term inhibition of L. monocytogenes	Ripened cheeses	[95]
L. lactis + E. durans	Used in biofilms over a wide temperature range	Significant reduction in L. monocytogenes in biofilms	Food processing environments (biofilms)	[96,97]
Probiotics (Bifidobacterium, probiotic yeasts)	General application; used in dairy and meat products	Inhibits L. monocytogenes; improves shelf life	Dairy (milk, cheese), meat	[98,99]
L. plantarum, L. sakei, L. rhamnosus	Various food products	Effective against L. monocytogenes	Dairy products	[104]
LAB in food packaging	Innovative packaging strategy	Prevents pathogen growth	General food applications	[109]
Probiotic metabolites (organic acids, EPS)	Under real processing conditions (to be evaluated)	Significant antimicrobial effects (potential)	General food products	[110]
Postbiotics (peptides, vitamins, fatty acids)	Used in films; encapsulated for better solubility	Demonstrated significant antibacterial activity	General food packaging	[110]
Nisin in processed cheese	Widely used; approved food additive (E234); GRAS; more effective at low pH	Effective against L. monocytogenes and S. aureus	Processed cheese	[112]
Nisin in queso fresco/fresh cheese	Neutral pH (above 6); high fat and calcium content reduce effectiveness	Ineffective inhibition of L. monocytogenes	Queso fresco, fresh cheese, whole milk	[113]
Nisin + hurdle technology	Combined with multiple mild treatments (e.g., low pH, refrigeration, salt)	Enhanced safety; reduced treatment intensity	General foods	[113]
Nisin (produced by L. lactis)	Nisin formation; thermal stability; resistant to digestion	Antimicrobial against Gram-positive bacteria	Meat, dairy, and aquatic products	[115]
Combined treatments (e.g., probiotics + UV or H2O2)	Requires screening to avoid probiotic inactivation	Potential synergistic antibacterial effects	General food matrices	[9]
Nisin + high-intensity ultrasound/UV-A light	Not specified	Enhanced antibacterial protection	General food	[118]
Encapsulated nisin	Long-term application	Altered sensory properties; efficacy under evaluation	Dairy (e.g., cheese)	[118]
Nisin + sesamol/carvacrol	Not specified	Synergistic effect; increased efficacy	Not specified	[118]
Nisin + lauric alginate + ε-polysine	28 days of cold storage	Effectively controlled L. monocytogenes	Fresh cheese	[118]
Nisin + phytic acid	Not specified	Increased efficacy against E. coli (Gram-negative bacteria)	Not specified	[119]
Bacteriocins (general, including nisin and pediocin)	Refrigerated storage for up to 12 weeks at 4 °C	Prevented L. monocytogenes growth	Meat, fish, dairy, salads, and juices	[120]
Liposome-encapsulated nisin	14 days at 7 °C	Reduced L. monocytogenes from 4.5 log CFU/mL to undetectable level	Whole and skim milk	[8]
Liposome-encapsulated P34 peptide	8 days	Controlled L. monocytogenes only in skim milk	Skim milk	[22]
Liposome-encapsulated sakacin + DOTAP	5 days at 7 °C	~5 log reduction in L. monocytogenes	Goat milk	[8]
Reuterin (8 AU/mL)	24 h at 37 °C	Completely inactivated L. monocytogenes	Milk	[121]
Reuterin + nisin	Not specified	Synergistic effect	Milk	[97]
Agilicin C7	Stable in various pH and solvents	Destroyed L. monocytogenes via membrane damage	Dairy and meat products	[97]
Bacteriocins + heat treatments	Lower temperature and duration	Preserved nutrients; improved efficacy	General food	[24,91]
Nisin-loaded zein microcapsules	Not specified	Reduced L. monocytogenes	Milk	[5]
Nisin in edible polymer films/coatings	Surface application	Prevented aggregation; maintained activity	Food surfaces (general)	[24,123]
Nanoencapsulated bacteriocins	Not specified	Improved antimicrobial activity	Food matrices (general)	[117,124,126,127]
Bacteriocins + probiotics/tea polyphenols/essential oils	Not specified	Effective in reducing L. monocytogenes	General food	[134]
Pediocin PA-1	Used in fermentation	Prevented contamination	Cheese (animal-derived foods)	[122]
Nisin + propolis	Not specified	Synergistic effect; preserved quality	Ice cream	[132]
LAB + grapefruit seed extract	Not specified	Effective in reducing bacterial growth	Fresh soft cheese	[132]
ListShield™ phage product	Applied on frozen foods	2.2 log reduction in L. monocytogenes	Prepackaged frozen foods	[130]
Listex™ P100 phage product	Applied on food and stainless steel	Reduced biofilms and contamination	Cheese, smoked fish, food surfaces	[130]
Phage endolysin PlyP100	Applied to queso fresco	Significant bacterial load reduction	Queso fresco	[132]

summarizes various treatment methods used to control L. monocytogenes in dairy products and presents key information, including the type of treatment, treatment conditions, inactivation efficiency, and the specific food matrix involved.

5. Natural Methods

Controversies surrounding synthetic additives and their effects on health have stimulated interest in the use of natural preservatives [135].

5.1. Essentials Oils

Essential oils are a promising alternative, having antimicrobial and antioxidant effects and being able to extend the shelf life of foods. However, the problems related to the low solubility in water, limited stability, and strong aroma of some essential oils can represent a disadvantage in certain food products. To overcome these limitations, oils can be integrated into edible and biodegradable films, which allow a controlled release of active compounds to create smart or active packaging. These represent innovative solutions that interact with the food atmosphere, allowing a more efficient preservation of food products. In addition, various modern technologies, such as hydrogels, nanoemulsions, and nanoparticles, are applied to improve the delivery of oils and protect foods against microbial contamination [136,137,138]. Also, microencapsulation of essential oils in a protective matrix, such as aloe vera or inulin, can improve the stability and antimicrobial and antioxidant efficacy of these oils during storage [139,140]. Natural methods and chemical agents used to reduce the incidence of L. monocytogenes in dairy products are presented in Figure 2.

In the food industry, essential oils are regulated and generally recognized as safe (GRAS), which has contributed to the growing interest in their use in food preservation. Although they are effective against bacteria and other microorganisms, the high concentrations required to ensure preservation can negatively influence the sensory properties of the products. Therefore, current research is focused on developing techniques that allow the use of essential oils at lower concentrations without compromising their antimicrobial efficacy [141,142].

Essential oils are volatile secondary metabolites extracted from various parts of plants, such as fruits, seeds, flowers, leaves, bark, and roots. They are bioactive components of spices and are formed by a complex mixture of volatile and non-volatile compounds, with a concentration ranging from 1% to 17%. Essential oils are mainly extracted from plants in the families Poaceae, Lamiaceae, Pinaceae, Rutaceae, Asteraceae, Lauraceae, Apiaceae, Piperaceae, Angiosperms, Sapindaceae, and Myrtaceae, among many others [143]. Essential oils are generally lipophilic and can be classified into various categories, such as monoterpenes, phenolic acids, alkaloids, flavonoids, carotenoids, and aldehydes. Their antimicrobial activities have been documented, including those of essential oils obtained from cinnamon, anise, dill, cloves, oregano, and zedoary. Synergistic effects of mixtures of these oils in combating microorganisms have also been reported [144].

The composition of essential oils varies depending on the part of the plant from which they are extracted by various methods, such as steam distillation, mechanical processing, or solvent extraction, and are composed of mixtures of phenylpropenes, terpenes, and other volatile compounds with strong antimicrobial and antioxidant properties. They can penetrate the bacterial membrane, inhibiting their essential functions [145]. For example, terpenes are known for their action against bacteria, viruses, and fungi. Currently, approximately 3000 essential oils are known, and some of them are commercially important, being used in the agronomic, cosmetic, chemical, perfume, pharmaceutical, and food industries due to their potential bioactivities [146].

Essential oils have the ability to act synergistically; that is, combining several essential oils can enhance antimicrobial activity, thus reducing the need for synthetic agents. These oils work by disrupting the cell membrane of microorganisms, making them effective against bacteria. Gram-positive bacteria are more susceptible to essential oils due to their cellular structure, while Gram-negative bacteria are more resistant [147].

Despite the success of in vitro tests, the practical application of essential oils in the food industry requires further research to determine the best methods of use, including in combination with other technologies, such as biodegradable packaging. Essential oils have been widely tested against bacteria, and studies have demonstrated their antimicrobial efficacy [148,149]. For example, cinnamon, thyme, and oregano essential oils have shown antilisterial activity, but their efficacy varies depending on the type of bacteria and the test conditions. Furthermore, these essential oils are more effective against Gram-positive than Gram-negative bacteria. Essential oils can also be used to prevent the formation of biofilms, a major problem in the food industry. However, their antilisterial effect needs to be studied more thoroughly under real-world conditions, such as low storage temperatures [22]. From a methodological point of view, essential oils can be integrated into various types of foods, such as cheese or minced meat, and their efficiency increases when used in nanoemulsions. Also, essential oils in the gaseous phase are promising for practical applications, such as salads. However, for the effective use of essential oils, it is important that they are compatible with the type of food to avoid altering the natural flavor of the product [21].

Edible films with essential oils are used to protect foods from external factors and microbial contamination. Polysaccharides, proteins, and lipids are used as biopolymers in the development of these films in combination with essential oils to provide antimicrobial activity. The addition of essential oils to such films can improve food preservation without affecting the sensory properties [137].

The use of essential oils in active packaging is an effective method for extending the shelf life of foods, but there is not enough information available about the commercial implementation of these solutions. A major challenge in using essential oils directly in foods is their volatility and instability under different environmental conditions. However, these limitations can be overcome by techniques such as encapsulation in nanoemulsions, which protect the essential oils and allow a controlled release of the active compounds [137].

Lemongrass essential oil (LEO) is one example of a natural compound with multiple applications, having antibacterial, antifungal, antioxidant, and anticancer properties. Due to this wide range of beneficial effects, LEO is widely used in the pharmaceutical, cosmetic, and food industries. However, its anticancer effects have not been fully studied in the human system, and further research is needed to confirm these properties [150].

The study by Moosavy et al. (2013) [151] demonstrated the efficacy of Mentha spicata essential oil in inhibiting L. monocytogenes in traditional Lighvan cheese. At concentrations of 2% and 2.5%, the oil was effective against the bacteria compared to the control group (p < 0.001) [151]. Eugenia caryophyllata was also effective against the microflora of refrigerated cheese, according to the study by Trajano et al. (2010) [152]. In addition, positive results have been reported for rosemary and thyme essential oils used in mozzarella cheese to inhibit the growth of bacteria such as L. monocytogenes. In another study, Moringa oil was tested to inhibit the growth of L. monocytogenes in various types of cheese [153]. Oregano and thyme essential oils have demonstrated potent antimicrobial activity against L. monocytogenes in feta cheese, extending the shelf life of the product. However, high concentrations of essential oil can affect the sensory properties of the products, so it is recommended to use lower concentrations to ensure food safety without compromising taste.

An important aspect of the use of essential oils is the assessment of the impact on the taste and aroma of food products. A study showed that Chamaemelum and Lavandula essential oils altered the sensory characteristics of yogurt in an unacceptable way. In contrast, the use of nanoencapsulated essential oils, such as Thymus capitatus, had a reduced impact on the sensory properties. Essential oils, such as those obtained from Thymus zygis, can replace synthetic preservatives, having significant antibacterial properties and potentiating the effect of other antimicrobial agents. Thus, essential oils can play an essential role in food preservation, providing a natural solution to reduce contamination and increase shelf life [137].

Essential oil nanoemulsions have been studied and applied in various food products. For example, a cumin oil nanoemulsion demonstrated antimicrobial activity against pathogens such as S. aureus and E. coli. This method of encapsulating essential oils in nanoemulsions and other nanostructured systems offers a promising solution for controlling microbial contamination in the food industry, including dairy products. However, scaling up these applications to a large scale still represents an economic challenge [8,154].

In recent years, research has shown that essential oils do not develop microbial resistance, which makes them particularly attractive to synthetic antimicrobials. Although essential oils have been extensively studied for their antimicrobial activity, there is little information on the comparison between the use of whole essential oils and their active components in combating microorganisms. Also, the effect of essential oils on biofilms and spores, more resistant forms of microorganisms, requires further investigation.

In addition to their antimicrobial properties, essential oils are also used to improve the flavor of food products, such as chocolate milk or cheeses. Added to these products, they play a crucial role in determining the intensity of the flavor, taste, and smell of the final product. However, the application of essential oils in dairy products is limited because the proteins and fats in these products can interact with them, reducing their antimicrobial effectiveness. For this reason, higher concentrations of essential oils are required to achieve the desired effects, which can negatively affect the sensory properties of the product.

A growing area of interest is the use of combinations of essential oils and biocontrol agents, such as bacteriocin-producing LAB, to improve food safety. These combinations have been shown to be effective in extending the shelf life of foods and controlling pathogenic microorganisms without affecting the sensory quality of the products [144].

For example, the combination of chitosan and caraway essential oil has been shown to be effective in improving the sensory properties of foods, while cinnamon essential oil has been successfully used in dairy products to enhance flavor and prevent oxidation. Recent studies have also shown that microcapsules of essential oils added to cheese can extend shelf life and inhibit bacterial growth [149].

Determining the optimal concentrations of essential oils that provide antimicrobial effects without negatively affecting sensory properties is essential for their practical application. Further research is needed to evaluate their effectiveness in combination with other preservation techniques and to explore the impact of essential oils on microorganisms in different states, including spores and biofilms [155].

Although there are many successful examples of the use of essential oils in food, challenges remain in finding the optimal balance between antimicrobial efficacy and maintaining sensory quality. The development of innovative solutions, such as nanoemulsions and the incorporation of essential oils into active packaging, could revolutionize the food industry and provide a natural and safe alternative to synthetic chemical preservatives.

5.2. Plant Extract

Plant extracts are derived from various parts of plants: leaves, flowers, roots, or seeds. Extraction methods vary, including cold pressing, steam distillation, hydrodistillation, supercritical CO₂ extraction, and the use of organic solvents. These methods influence the chemical composition and biological effects of the extracts [156].

Plant extracts and essential oils are effective against Gram-negative and Gram-positive bacteria and are increasingly being studied for their use in the food industry. In particular, essential oils from spices such as pepper, ginger, garlic, and onion have demonstrated antimicrobial and antifungal activities, making them promising candidates for the development of new natural antimicrobial agents [145]. Plants of the genus Thymus, used both in traditional medicine and as spices, have been studied for their antimicrobial potential. Thymus zygis has shown broad antimicrobial activity, indicating great potential for food preservation [145].

In parallel, the use of synthetic compounds such as aromatic hydrocarbons, benzimidazoles, and synthetic antioxidants (BHA, BHT) raises concerns about long-term food safety due to potential carcinogenic effects. In response, consumers prefer natural additives, and plant products are becoming viable alternatives to synthetic preservatives. Essential oils and plant extracts offer promising solutions for extending the shelf life of perishable foods, especially in the case of fresh, minimally processed, or ready-to-eat products. Plant antimicrobials are composed of secondary metabolites, such as phenolic compounds, terpenes, and alkaloids, which act on microbial cells. Although plant antimicrobials are effective, their use in the food industry is limited by chemical instability, low dispersibility, and flavor issues. In this context, various stabilization techniques, such as nano-emulsions and encapsulation, improve the activity and controlled release of antimicrobial compounds during food storage. A great diversity of structures among plant secondary metabolites (PSM) occurs in nature (e.g., over 12,000 known alkaloids, over 10,000 phenolic compounds, and over 25,000 different terpenoids). Structurally, plant antimicrobials can be divided into two classes: PSMs with one or more nitrogen atoms in their structures, such as alkaloids and glucosinolates, and PSMs without nitrogen, such as terpenoids and phenolics. Alkaloids, glucosinolates, and phenolics are water-soluble compounds, while terpenoids are lipophilic PSMs [134].

The use of garlic extract encapsulated in nanoliposomes showed increased efficacy against Listeria spp. in milk, significantly reducing the number of viable cells after a few hours of incubation. Co-encapsulation of garlic extract with nisin further improved their efficacy, with significantly better results in reducing the population of L. monocytogenes, S. aureus, Salmonella, and E. coli in milk. This technique was also effective at low temperatures, maintaining a significant reduction in pathogenic bacteria for up to 25 days.

Extracts of cinnamon, garlic, rosemary, and oregano have been shown to be effective in inhibiting L. monocytogenes in various types of cheese. Plant extracts and essential oils are well-received in the dairy industry, contributing to the improvement of flavors and antioxidant and antibacterial properties. They can be added in free or encapsulated form to protect the active compounds and control their release. Plant extracts and essential oils can also support the survival of probiotics and inhibit pathogenic microorganisms, increasing the value of dairy foods [157].

Natural fruit preservatives, such as citrus or apple peel extracts, have been used to prevent spoilage of milk and dairy products, demonstrating efficacy against lipid oxidation and microbial growth. For example, apple peel extracts added to yogurt improved its properties without negatively affecting pH or syneresis. In the case of cheese, the use of blueberry extracts improved appearance and inhibited microbial growth, while pomegranate peel extract increased the sensory scores of the treated cheese.

Grapefruit extract was used in a biodegradable polybutylene adipate-co-terephthalate (PBAT) packaging, combined with grapefruit seed extract (GSE), to inhibit L. monocytogenes in soft cheese. Supplementation with kimchi-isolated bacteria, such as Leuconostoc mesenteroides and Lactobacillus curvatus, improved the efficacy of the extract in controlling the bacteria. Thus, the PBAT and grapefruit seed extract film combined with L. mesenteroides provided better results than the separate application of these agents, suggesting that a combined approach provides superior pathogen control. After the application of the biological agents, surviving bacteria can grow at low temperatures, with accelerated recovery at higher temperatures.

5.3. Organic Acids

Organic acids, such as lactic, malic, citric, and acetic acids, are commonly used to prevent microbial contamination of foods. They inhibit microbial activity by lowering pH, reducing enzyme activity, and causing cell damage. Pintado et al. (2009) [158] demonstrated that a combination of organic acids (lactic, malic, citric) with nisin had significant antilisterial efficacy against L. monocytogenes isolated from cheeses. A novel method of using organic acids involves their incorporation into edible coatings. For example, calcium alginate gels with lactic and acetic acids or chitosan with citric, acetic, and malic acids have been shown to be effective against L. monocytogenes in various food products [159].

5.4. Macroalgae

Macroalgae represent a promising source of natural biopreservatives due to their antimicrobial potential and contribution to improving the quality of food products while extending their shelf life [156].

Various methods have been used to reduce the incidence of L. monocytogenes in dairy products. All these methods have advantages but also limitations, which are presented in

Table 3. Advantages and disadvantages of some methods used to reduce the incidence of L. monocytogenes in dairy products.

Identified Method	Advantages	Disadvantages	References
Conventional thermal methods	Destroys contaminating microorganisms and inactivates enzymes	Reduce the sensory and nutritional qualities of the finished product	[13,30]
Direct steam injection	It is considered one of the best technologies to prevent thermal deterioration of milk	High cost and increased complexity	[48]
High-pressure processing (HPP)	More effective method in liquid foods than in solid foods Negligible effects on health and organoleptic and nutritional properties of food products	May cause incomplete microbial inactivation	[1,63,160]
Ionizing radiation	Cheese decontamination Use for treating packaged foods to reduce the risk of post-processing contamination	May cause incomplete microbial inactivation A high dose of irradiation may cause product discoloration and unpleasant odors	[161,162,163,164]
Ultrasounds	Efficient, cost-effective, and environmentally friendly process Extending the shelf life of finished products	Prolonged exposure can develop a metallic, burnt taste and rubbery appearance in the milk	[10,68]
LAB	Produce bacteriocins Minimally processed food products and better nutritional and sensory value Shortens the ripening process of cheeses Extends the shelf life of finished products Resistance to acids	In vitro antilisterial activity must be demonstrated in food applications Only two genera of LAB (Lactobacillus and Lactococcus) are considered GRAS More strains of LAB should be used for more effective control	[7,22,81,89,91,92,98,107,165]
Probiotics	Maintaining the initial physicochemical properties of food products Improving organoleptic properties Extending the shelf life of finished products	Antilisterial activity must be demonstrated when obtaining food products May cause some metabolic disorders or the generation of biogenic amines	[166,167]
Bacteriocins Bacteriolysins	Directly disrupts the integrity of the bacterial membrane, producing inactivation of the bacterial cell High efficiency and convenience Active over a wide pH range, resistant to high temperatures Extends the shelf life of finished products Provides additional protection in high-temperature processes Prevents loss of organoleptic and nutritional properties In situ production (protective cultures)	Poor solubility, uneven distribution High cost Limited antilisterial activity Inactivation by other food additives Efficacy may be affected by various environmental factors, e.g., storage temperature Sensitivity to the presence of proteolytic enzymes	[9,10,24,87,90,124,125,127,132,168]
Bacteriophages	High specificity towards the pathogen Does not affect the beneficial natural microflora, organoleptic qualities, or nutritional value Easy to isolate and propagate Destroys biofilms Safe for humans and the environment	Multiple bacteriophages may be required if food is contaminated with multiple bacterial pathogens Sensitive to commonly used disinfectant Negative consumer perception of the use of “viruses” in food	[3,17,129,130,133]
Natural antimicrobials	They enhance the flavor of food, improve nutritional value, and bring health benefits Maintaining the original physicochemical properties of food products	Undesirable organoleptic properties at high concentrations Chemical instability Limited availability Limited dispersibility	[8,142,157,169]
Essential oils	Extending the shelf life of finished products Natural flavoring agents Improving the functional and sensory properties of dairy products	Production of strong flavors that may be undesirable flavors in some food products Less effective in controlling L. monocytogenes	[136,138,140,143,144,148,149,155]
Chemical agents	Significantly reduce the adhesion of L. monocytogenes to the surface of food products	Negative health effects (allergic or carcinogenic)	[26,99]
Mechanical–bactericidal (MB) surfaces	High biofilm control potential	Challenges related to scalability, cost-effectiveness, mechanical and chemical durability, and complex composition of food products	[166]

.

6. Use of Chemical Agents

Antimicrobials are substances that inhibit or kill microorganisms, including both bacteriostatic (prevent growth) and bactericidal (kill microorganisms) agents. In the United States, regulations for antimicrobials vary depending on how they are used.

The differences between disinfectants and antibiotics are significant. Antibiotics are chosen based on the susceptibility of microorganisms, and their action targets specific components of the cells, which can lead to resistance by modifying these components. Disinfectants, on the other hand, are selected based on their intended use, interaction with materials, and cost. Unlike antibiotics, some disinfectants contain one or more active ingredients. Advice on their selection is usually provided by disinfectant suppliers.

Food safety practitioners have the responsibility of translating scientific research into effective industrial practices. A major challenge is a terminological confusion, an example of which is the different interpretations of the term “disinfectant resistance”. In the scientific literature, it may describe the ability of a bacterium to survive sublethal doses of disinfectant, while the industry may understand that the product is ineffective even at maximum concentrations. Practitioners need to understand and clearly communicate these nuances, especially in facilities producing ready-to-eat (RTE) foods, where cleaning and sanitation are essential for the control of L. monocytogenes and other pathogens [170]. To the best of our knowledge, true resistance to disinfectants has not been observed in L. monocytogenes at recommended concentrations and conditions of use [171].

Hygienic design of equipment and facilities, as stipulated in international regulations, is crucial for the effectiveness of cleaning. Smooth, non-absorbent, and easy-to-clean surfaces are essential to prevent contamination with microorganisms. Hard-to-reach equipment, worn materials, and porous surfaces are hiding places for pathogens, including L. monocytogenes. Cleaning of components that do not come into direct contact with food is also important, as they can serve as sources of contamination [172].

Disinfectant rotation is a practice used to prevent the development of antimicrobial resistance in food processing facilities. It involves alternating disinfectants with different active ingredients to reduce the risk of microbiome adaptation.

Although this practice is recommended by the FDA (2017) for the control of L. monocytogenes in ready-to-eat (RTE) foods, scientific support for disinfectant rotation remains limited. The available studies are mostly guidelines documenting common practices, but there is no clear evidence that rotation is essential for food safety [173]. Although disinfectant rotation is not harmful, its implementation as an essential practice without a solid scientific basis can lead to unnecessary complications. More research is needed to evaluate the effectiveness of this method and the long-term impact of microbial tolerance to disinfectants [174].

In the food industry, cleaning and sanitizing are essential for the removal of microorganisms and soil from surfaces, equipment, and food contact areas. Various chemicals, such as chlorine, chlorine dioxide, hydrogen peroxide, quaternary ammonium compounds, ozone, and others, are used in these processes [175]. Aqueous chlorine is effective in controlling microbial growth, but its activity decreases under alkaline conditions, forming toxic substances such as chloramines and trihalomethanes (THMs). In this context, chlorine dioxide (ClO₂) is a superior alternative, having double the oxidative power and being more effective under alkaline conditions and in the presence of organic compounds. Research shows that ClO₂ in the gaseous state has greater penetrability, being more effective in eliminating microorganisms hidden in surface irregularities and biofilms [176].

Trinetta et al. (2013) [177] evaluated the efficacy of ClO₂ at high concentrations for short periods in the treatment of fresh produce, suggesting that it could be used on a large scale for product sanitation. Luu et al. (2021) [178] demonstrated that ClO₂ gas treatment at low concentrations can effectively reduce L. monocytogenes, indicating its potential as a disinfectant in food processing. However, the industrial-scale use of this disinfectant could be limited by cost and operational constraints.

7. Other Methods for Reducing L. monocytogenes

Recently, research has focused on antibacterial solutions based on solid surface engineering, which reduce biofilm formation through antifouling or destruction mechanisms. These solutions aim to interfere with the early stages of biofilm development, thereby preventing its maturation and persistence. Advances in nanofabrication and nanoengineering have enabled the development of nanostructured surfaces inspired by nature that prevent microbial invasion through physical disruption rather than chemical effects. However, the application of these mechano-bactericidal (MB) surfaces in the food industry is hampered by challenges such as scalability, cost, durability, and complexity of food composition. MB surfaces offer a promising approach to combat biofilms, as their physico-mechanical inactivation mechanisms are different from those of traditional biocide-based disinfectants.

It is necessary to transfer the results obtained in the laboratory to real-world applications with direct benefits on human life and food safety to realize the full potential of MB surfaces, which could significantly contribute to improving food safety and quality, in combination with other measures to prevent microbial contamination [166].

8. Conclusions

L. monocytogenes is a Gram-positive, highly pathogenic bacterium known to contaminate food, especially unpasteurized dairy products. Having become a major concern since the 1980s, it can survive in extreme conditions (low temperatures, high salinity, variable pH) and forms disinfectant-resistant biofilms in food processing environments. Its persistence is associated with poor hygiene, ineffective cleaning protocols, and insufficient staff training, with biofilms providing additional protection against environmental factors and antimicrobial agents.

Nonthermal sterilization technologies in the food industry include chemical sterilization, irradiation, high-pressure processing (HPP), ultrasound, and ozonation. These methods offer advantages like nutrient and flavor retention but also face challenges like high costs and variable efficiency. The combination of these technologies, known as “multi-barrier technology”, is a promising solution for increasing food safety and extending product shelf life. In the dairy industry, lactic acid bacteria (LAB) and bacteriocins offer multiple advantages in terms of food safety and product quality. Bacteriophages offer a safe and effective alternative to traditional preservation methods, but more research and regulations are needed. Natural preservatives, including essential oils, offer multiple benefits, including antimicrobial and antioxidant activities. These technologies are continually being explored and improved to meet modern consumer demands and expand their use globally. With appropriate regulations and technological innovations, essential oils could become an essential component of the future food industry.

The dairy industry continues to explore new technologies and methods to meet the demands of modern consumers, and bacteriocins produced by lactic acid bacteria are an essential tool in this endeavor. At the same time, research continues to improve the effectiveness of bacteriocins and expand their use globally. Bacteriophages offer a promising solution for combating food pathogens, providing a safe and effective alternative to traditional preservation methods. Although more research and clearer regulations are still needed in some regions, phage biocontrol is an innovative and effective approach to food safety. The use of natural preservatives, including essential oils, represents a viable alternative to synthetic additives, with multiple benefits, including antimicrobial and antioxidant activities. However, their effective application in the food industry requires further research, especially to determine the best ways to integrate them into products and to ensure compatibility with the sensory characteristics of foods.

In conclusion, continued research is needed to optimize control and reduction strategies for L. monocytogenes to ensure a balance between food safety and product quality.