Several scientists have questioned the relationship between the persistence of
L. monocytogenes and its ability to form biofilms
[22][23]. However, both concepts are not fully understood and require more in-depth discussion. Biofilms have been defined as the population of microbial cells adherent to each other and/or to the surfaces by producing a three-dimensional extracellular matrix
[24][25]. The formation of biofilms occurs in sequential steps
[26][27]: (1) attachment of planktonic bacteria to solid surface by electrostatic forces, van der Waals forces, and hydrophobic interactions, (2) proliferation of cells and formation of extracellular polymeric substances, (3) construction of multilayer cellular clusters with channels for the flow of nutrients and waste, and (4) biofilm formation and cell dispersion for subsequent colonization on other surfaces.
L. monocytogenes is able to adhere to a variety of surfaces, including stainless steel, glass, propylene, rubber, quartz, marble, granite, and food surfaces such as chicken skin and beef surfaces
[28][29][30].
L. monocytogenes in biofilms is protected from a variety of environmental stresses, such as UV light, desiccation, acids, and toxic metals, and may survive antimicrobial and sanitizing agents such as iodine, chlorine, and quaternary ammonium compounds
[31]. For example, Russo et al. (2018) found that sodium hypochlorite (200 ppm,
v/
v), hydrogen peroxide (2%,
v/v), and benzalkonium chloride (200 ppm,
w/
v) were not able to completely eradicate established biofilms in experimental conditions
[31]. The study also suggested that subminimal concentrations of antimicrobial and sanitizing compounds may encourage the growth of the resistant population of
L. monocytogenes. Some studies have shown that persistent strains show biofilm formation
[23], while other studies have found no relationship between persistence and biofilm formation
[22].
3. Novel Approaches to Control of L. monocytogenes
Different listericidal and listeriostatic approaches can be applied to control
L. monocytogenes in foods and food production environments. Several conventional approaches, such as pasteurization, sterilization, freezing, chilling, acidification, fermentation, drying, filtration, antimicrobial agents, and additives have been used to control
L. monocytogenes growth in foods. However, some of these approaches are harsh and adversely impact foods’ nutritional and sensory attributes
[52]. In recent years, consumers have become more interested in buying food products that are minimally processed, free of additives, shelf-stable, and have a better nutritional and sensory value
[53]. In order to fulfill these requirements, several novel control strategies have emerged recently.
Figure 1 shows some novel approaches to control
L. monocytogenes in foods and food production environments, based on thermal, non-thermal, biocontrol, natural, and chemical methods. Generally, a single approach is not effective in controlling
L. monocytogenes, and a combination of approaches is required, also known as hurdle technology. For example, irradiation can be an effective approach to controlling
L. monocytogenes in foods. However, some cells may survive and grow even after irradiation; hence, using an antimicrobial agent after irradiation may further suppress the growth of
L. monocytogenes [54]. This deliberate and judicious combination of control strategies to create a series of hurdles that a microorganism is not able to overcome is called hurdle technology. Several studies have evaluated different hurdle strategies to control
L. monocytogenes in food products. For example, Upadhyay et al., 2014 evaluated a combination of four plant-derived antimicrobial compounds (carvacrol, thymol, b-resorcylic acid, and caprylic acid), along with H
2O
2 and high-temperature treatment to control
L. monocytogenes in cantaloupes
[55]. In another study, Espina et al., 2014 applied a combination of pulsed electric field (PEF), mild heat, and natural essential oils to inactivate
L. monocytogenes in liquid whole egg
[56]. The study found that a combination of these techniques was as effective as ultra-pasteurization for killing
L. monocytogenes but with a less detrimental impact on the product’s sensory attributes
[56]. Combining two or more approaches can produce a synergistic effect by hitting different targets that disturb the homeostasis of microorganisms
[57]. Several studies have successfully applied different combinations of conventional and innovative strategies to control
L. monocytogenes in foods and food production environments
[58][59][60][61][62].
Figure 1. Novel approaches to control L. monocytogenes in food products and food production environments, categorized as thermal methods, non-thermal methods, biocontrol methods, natural methods, and chemical agents.
3.1. Thermal Methods
Conventionally, thermal processing methods such as pasteurization and sterilization are applied to control
L. monocytogenes in foods. Direct hot air, steam, heat exchangers, and hot water baths are commonly used for the thermal processing of foods at different temperature-time combinations
[63]. The temperature for pasteurization ranges from 60 to 80 °C to kill microorganisms and inactivate enzymes, whereas the temperature for sterilization is >100 °C to kill spores and spore-forming bacteria
[64]. D-value is the heating time required to kill 90% of microorganisms or to reduce the microbial concentration by one log. The thermal resistance of microorganisms increases with an increase in the D value
[53]. For example, the D-value of
L. monocytogenes present in milk samples ranged from 1683.7 s to 0.7 s when milk samples were heated from 52.2 to 74.4 °C
[65]. Post-package pasteurization of ready-to-eat foods is gaining recognition as a useful technique to reduce the risk of post-processing contamination of
L. monocytogenes in food products
[66][67]. Post-package pasteurization or sterilization is done by packing food in the container and heating the container using steam or hot water in a retort, such as a pressure cooker or autoclave
[53]. However, not all food products are suitable for high-temperature treatments as they may reduce the organoleptic quality of the product.
Several novel interventions have emerged for precise and rapid heating of foods while maintaining their sensory and nutritional characteristics, such as microwave, radio frequency, ohmic heating, and direct steam injection. In the electromagnetic spectrum, both microwave and radio frequencies belong to non-ionizing radiation, with radio frequencies ranging from 30 to 300 MHz and microwaves ranging from 300 MHz to 300 GHz
[68]. The primary mechanism involved in heating with microwave and radiofrequency is dielectric heating, which is based on the interaction of molecules with dipolar nature (e.g., water) and ionic charges in foods with electromagnetic radiation oscillating at a very high frequency
[68]. The dielectric system provides non-contact, uniform, and volumetric heating of the product and has been extensively evaluated for its thermal and non-thermal antimicrobial activity. Sung and Kang, 2014 assessed the effectiveness of microwave heating for the inactivation of
L. monocytogenes and other pathogenic microorganisms in salsa products
[69]. The study found that microwave heating at 915 MHz could be an alternative to pasteurization, as it can kill microorganisms while maintaining the overall quality of the product
[69]. Microbial destruction by microwave occurs by denaturation of cellular protein structure, causing rupturing of the cell membrane
[53]. Awuah et al., 2005 evaluated the application of radiofrequency in inactivating
Listeria in milk and found up to 5-log reduction at 1200 W, 65 °C, and 55.5 s
[70]. Radiofrequency and microwave have been applied to control microorganisms in products such as fruit juices, meat products, ready-to-eat products, coconut water, catfish, eggs, and pasta products.
3.2. Non-Thermal Methods
High-pressure processing (HPP) is a novel non-thermal method where a high pressure above 100 MPa is applied to the product using pressurized liquid such as water. HPP causes the inactivation of microorganisms through the mechanism of denaturing cell membrane, unfolding protein structure, changing cell membrane fluidity, ribosome dissociation, leakage of intracellular components, and eventually cell disruption
[71]. The effectiveness of HPP in the inactivation of
L. monocytogenes depends on parameters such as temperature, applied pressure, holding time, and properties and composition of the food matrix. For example, HPP is a more effective technique in the inactivation of
L. monocytogenes in liquid foods than solid foods
[72]. HPP may not always cause microbial inactivation and may sub-lethally damage the microbial cells, which may recover later. Therefore, combining HPP with other hurdles can effectively kill
L. monocytogenes, for example, Nassau et al., 2017 evaluated the effectiveness of a combination of endolysin with HPP to inactivate
L. monocytogenes in a buffer and found that a combination of two techniques could result in a 5-log reduction of
L. monocytogenes cells
[59]. The study indicated that HPP, when applied individually at 300 MPa for 1 min at 30 °C, could reduce the cell count by only 0.3 log CFU, but when applied in combination with endolysin, it could cause an effective inactivation of
L. monocytogenes even at lower pressure levels.
Another alternative non-thermal method for controlling
L. monocytogenes is the pulsed electric field (PEF), wherein the inactivation of microorganisms takes place by using high electric field pulses (>18 kV/cm) for a short time
[53]. A high voltage pulsed electric field disrupts the bacterial cell membrane, leading to releasing intracellular components and eventually killing the microorganism. Several factors influence the inactivation kinetics of PEF, including electric field strength, pulse length, pulse number, temperature, pH, and conductivity
[73]. Gómez et al., 2005 assessed the effectiveness of PEF on the inactivation of
L. monocytogenes in media of different pH (3.5–7.0) and found that PEF was more effective at lower pH and higher electric field strengths
[74]. At pH 3.5, a treatment of 28 kV/cm for 400 μs was able to reduce 6.0 Log
10 cycles of
L. monocytogenes cells
[74]. In recent years, PEF technology has been explored for controlling
L. monocytogenes in various food products such as milk and dairy products, juices, and soups.
Ultrasound is another emerging non-thermal technology for processing food products. High powered ultrasound with a frequency of 20 to 100 kHz is used in food production environments to kill microorganisms through a mechanism called cavitation
[75]. In this mechanism, gas bubbles are formed in the liquid medium as a result of sonication, the bubbles expand until a critical point is reached where ultrasonic energy is insufficient to retain the vapor phase of the bubbles, hence the bubbles become unstable and collapse, creating shock waves that damage the bacterial cell wall. Baumann et al., 2009 evaluated the efficacy of ultrasound for the removal of
L. monocytogenes biofilms from stainless steel chips and found that power ultrasound (20 kHz, 100% amplitude, 120 W, 60 s) was effective in reducing recoverable cells (3.8 log CFU/mL reduction)
[76]. When ultrasonication was combined with ozonation (ozone concentration 0.5 ppm), there were no recoverable cells after the treatment (reduction of 7.31-log CFU/mL)
[76]. Ultrasound is an effective technology in food processing and inactivating microorganisms, but it may impact the quality of food products by creating free radicals, off-flavors, and changing the composition of the food matrix.
Ionizing irradiation involves exposing food to radiation which causes ionization on interaction, such as gamma rays (
60Co and
137Cs), high energy electron beam, or X-rays. Irradiation can be used for the decontamination of products such as meat, poultry, egg products, fish products, and spices
[77]. A study by Bari et al., 2005 indicated that a low dose of ionizing irradiation can be effective in reducing
L. monocytogenes on fresh vegetables without significantly changing the color, texture, taste, and appearance of the product
[78]. Irradiation in frozen foods allows a higher dose level before developing off-flavor, for example, in frozen poultry, the dose level can be at least two times higher as compared to chilled poultry
[79]. In a study by Velasco et al., 2015, electron beam was applied to eliminate
L. monocytogenes from soft cheeses
[80]. The study found that irradiation was able to reduce the bacterial load, but injured cells were recovered during storage. Combining irradiation with other hurdles can be more effective compared to irradiation alone. For example, Mohamed et al., 2011 found that a combination of gamma radiation and nisin could be effective in eliminating
L. monocytogenes in meat products
[81]. One advantage of irradiation is that it can be used to treat food in packages to reduce the risk of post-process contamination. An irradiation dose of 30–50 kGy is used to reduce microbial contamination of foods called as radappertization
[82]. A high irradiation dose may cause discoloration of the product, and release of radiation-induced off-odors and off-flavors during storage. Therefore, it is important to carefully determine the dose level and apply the minimum possible doses to achieve desired level of control.
Ultraviolet radiation has germicidal effect and is used to eliminate microbial load on surfaces, air, water, and is now approved for microbial reduction in foods and juices
[83]. UV-C light (254 nm) is absorbed by most microorganisms which leads to alteration in microbial DNA by dimer formation, limiting the ability of microorganism to multiply and grow. Adhikari et al., 2015 evaluated the effectiveness of UV light for the inactivation of
L. monocytogenes on fruit surfaces and found a higher inactivation on fruits with a smoother surface, such as apples (1.6 log CFU/g reduction at 3.75 kJ/m
2), as compared to fruits with a rough surface such as cantaloupe (1.0 log CFU/g reduction at 11.9 kJ/m
2)
[83]. Kim et al., 2002 observed a >5 log reduction of
L. monocytogenes on stainless steel after treating with UV-C (500 μW/cm
2) for 3 min
[84]. Similar observations were made by Sommers et al., 2010, indicating the efficacy of UV-C light for routine decontamination of food-contact surfaces
[85].
3.3. Biocontrol Methods
Biocontrol methods against
L. monocytogenes include bacteriophages, bacteriocins, and competitive bacteria. Bacteriophages are viruses that infect and kill bacteria for propagation. Bacteriophages are highly specific toward their target bacteria and have no detrimental effect on non-target microbes, which is considered a vital advantage for biocontrol specificity and sensitivity
[86]. Bacteriophages can undergo two types of life cycle: lytic and lysogenic. In the lytic cycle, bacteriophage attaches to the bacterial cell, introduces phage DNA into the bacterial cell, utilizes bacterial machinery to encode and assemble new phage particles, and at the end, releases progeny phage into the environment by lysing the bacterial cell. In the lysogenic cycle, the phage genome is integrated into the host DNA and replicates with bacterial DNA. The lysogenic phages (also known as temperate phages) continue to replicate with the host cell until an unfavorable condition occurs, which initiates the lytic cycle. Temperate phages are not suitable for biocontrol as they may not result in host cell death, whereas lytic phages are considered suitable for biocontrol due to their virulence. Bacteriophages are not considered a risk to humans upon consumption due to their high specificity towards host bacterial cells, and consequently, some commercially produced bacteriophages have been recommended as GRAS (generally recognized as safe) by FDA, such as ListShield
TM and Listex
TM P100
[86]. Gutiérrez et al., 2017 evaluated the effectiveness of ListShield
TM and Listex
TM P100 against
L. monocytogenes in biofilms and Spanish dry-cured ham
[86]. The study found that both products effectively removed 72-h old biofilm from stainless steel surface after four-hour treatment at 12 °C. Application of ListShield
TM on Spanish dry-cured ham was effective in lysing 100% of strains examined, whereas Listex
TM P100 was effective in lysing 64% of strains. The study suggested that these phage-based products can be useful for biocontrol of
L. monocytogenes in food production environments. Phages can be applied to food products using different methods, such as spraying and dipping, or by using novel approaches such as immobilization on inert surfaces
[87]. An alternative to using whole bacteriophages to control
L. monocytogenes can be the application of endolysins. Endolysins are hydrolytic enzymes encoded by phage genome towards the end of lytic cycle to break the bacterial cell wall and release the progeny phages. Endolysins can be recombinantly produced and applied externally to bacterial cells without requiring bacteriophages. Ibarra-Sánchez evaluated the effectiveness of endolysin PlyP100 to control
L. monocytogenes in Queso Fresco and compared it with nisin
[88]. According to the study, PlyP100 showed bacterial reduction at varying
L. monocytogenes inoculum levels, and showed no recovery at inoculum level of 1 log CFU/g. The endolysin was stable for 28 days and showed consistent antilisterial action. Nisin was not as effective as PlyP100 to control
L. monocytogenes, however, a combination of the two showed a strong effect with no countable
L. monocytogenes cells after 4 weeks of refrigeration
[88].
3.4. Natural Methods
The application of natural and plant-derived antimicrobials, such as spices, herbs, essential oil, plant extract, and organic acids, is gaining attention for food preservation as an alternative to chemical preservatives. Natural antimicrobials have several benefits in addition to inhibiting microorganisms, for example, they increase flavor in the food, improve fragrance, improve medicinal value, and improve the nutritional quality of food. Spices and herbs are derived from different parts of the plants, such as clove from flower bud which contains antimicrobial compound eugenol, cinnamon from bark which contains cinnamic aldehyde, turmeric from rhizome which contains curcumin, mustard from seeds which contains allyl isothiocyanate, and thyme and oregano from leaves which contain thymol and carvacrol
[89][90]. Numerous studies have evaluated the antimicrobial activities of spices and herbs against
L. monocytogenes. Ting and Deibel, 1991 examined 13 species against
L. monocytogenes and found that cloves had bactericidal effect and oregano had bacteriostatic effect at 0.5% or 1% concentration at 4 °C and 24 °C
[89]. When tested against meat slurry, a 1% concentration of clove or oregano did not have much inhibitory impact on
L. monocytogenes [89]. Essential oils are aromatic, volatile oils obtained from different parts of the plants, including flowers, leaves, seeds, buds, roots, bark, woods, fruits, and peels. They are extracted by pressing and distillation or supercritical fluid extraction. Antimicrobial activity of essential oils is due to the presence of different compounds such as terpenes, phenolic compounds, aldehydes, and esters, most of which are classified as GRAS. Studies have indicated that phenolic compounds in essential oils cause a change in the permeability of bacterial cell membrane, intervene in ATP (Adenosine 5′-triphosphate) formation, and disrupt proton motive force
[90]. Several different essential oils have been evaluated for their effectiveness against
L. monocytogenes. Sandasi et al., 2007 examined the effectiveness of five common essential oils (α-pinene, 1,8-cineole, (+)-limonene, linalool, and geranyl acetate) against biofilms
[91]. Morshdy et al., 2021 examined essential oils (cinnamon bark oil, thyme oil, coriander oil, lavender oil, rosemary oil) against
L. monocytogenes isolated from fresh retail chicken meat and found that cinnamon bark oil showed the highest antilisterial activity
[92]. Studies have indicated that gram-positive bacteria are more sensitive to essential oils
[90]. Essential oils due to their hydrophobic nature can easily pass through the cell wall of gram-positive bacteria, whereas the outer membrane of gram-negative bacteria possesses hydrophilic nature and limits the diffusion of essential oils
[90]. One main disadvantage of using essential oils as antimicrobial agents is the production of strong aromas and off-flavors that can be undesirable in some food products.
3.5. Chemical Agents
In food production facilities, cleaning and sanitation are important steps to eliminate microorganisms and dirt from food-contact surfaces, equipment, floors, and walls. Various chemical agents, such as chlorine, chlorine dioxide, hydrogen peroxide, quaternary ammonium compounds, ozone, nitrites, and phosphates are used for cleaning and sanitation. Aqueous chlorine is an effective agent to control microbial growth; however, its antimicrobial activity decreases in alkaline conditions and leads to the formation of toxic reaction products such as chloramines and trihalomethanes (THMs)
[93]. Chlorine dioxide (ClO
2) is used as an alternative to chlorine, as it is more potent (~2 times oxidation capacity) in killing bacteria and is not affected by alkaline conditions and organic compounds. Researchers have investigated the application of ClO
2 gas to disinfect several food products and food-contact surfaces
[94][95][96]. ClO
2 has more penetrability than aqueous ClO
2 and has a better reach to microorganisms hidden in surface irregularities and biofilms
[94]. Trinetta et al., 2013 evaluated the effectiveness of high-concentration short-time ClO
2 for treating fresh produce and suggested it can be a useful technique for sanitizing produce in large-scale operations
[97]. Luu et al., 2021 indicated that treatment with ClO
2 gas (<5 mg/L) in gas permeable sachets could effectively reduce
L. monocytogenes on strawberries and blueberries
[98]. Results suggest that ClO
2 gas has potential as a sanitizer for food processing; however, there can be economic and operational constraints in using this method on a large scale.