Exposure to cold temperatures following harvest in order to minimize and/or inhibit the effects of wounding stress is recognized as one of the principal factors controlling the quality of fresh-cut leafy vegetables [48,49]. Although 0 °C is usually the desirable temperature for most fresh-cut products, in practice many of them are shipped and marketed at temperatures ranging from 5 to 10 °C [50].

In response to temperature downshift, a number of changes take place in prokaryotic cellular physiology such as, (i) decrease in membrane fluidity, (ii) stabilization of secondary structures of nucleic acids leading to reduced efficiency of mRNA translation and transcription, (iii) inefficient folding of some proteins, and (iv) hampered ribosome function [51]. A number of cold shock proteins are induced to cope with these harmful effects of temperature downshift. For all organisms, maintenance of functional cell membranes is a limiting factor for survival. Upon cold shock the physical status of biological membranes is altered from being fluid to becoming rigid. In a process generally termed homeoviscous adaptation, with decreasing temperature, bacteria incorporate fatty acids of lower melting points into lipids in a species-specific mode to re-establish membrane integrity and hence function. In this picture, the introduction of double bonds into acyl chains can either be achieved anaerobically during fatty acids synthesis or aerobically by modification of readily synthesized fatty acids through fatty acid desaturase enzymes. Transcription and translation are closely coupled in bacterial cells. However, transcription machinery and ribosomes generally occupy different subcellular regions in bacteria such as Escherichia coli and Bacillus subtilis, indicating the need for (a) mechanism(s) coupling these processes. A prime function of this mechanism(s) would be ensuring the transfer of unfolded mRNA from the nucleoid to the ribosomes, which need linear mRNA for the initiation of translation. During conditions of a sudden decrease in temperature (cold shock), secondary structures in mRNA would pose an even greater problem for the initiation process. Two conserved classes of proteins, cold shock proteins (CSPs) and cold induced RNA helicases (CSHs), appear to be key players in the prevention of secondary mRNA structures and in transcription/translation coupling. CSPs are general mRNA-binding proteins, and like CSH-type RNA helicases, the presence of at least one csp gene in the cell is essential for viability [52]. E. coli contains nine CSPs (CspA to CspI), of which four (CspA, -B, -G, and -I) are cold shock inducible [53]. CspA, CspC, and CspE are RNA-binding proteins which function as transcriptional antiterminators by preventing the formation of secondary structures in the nascent RNA. Csp-induced transcriptional antitermination is responsible for the increased expression of several genes [54,55]. B. subtilis contains three CSPs (CspB, -C, and -D); CspB is essential for cellular growth in a strain lacking CspC and CspD and plays an important role for efficient protein synthesis at optimal and low temperatures [56], while CspB and CspC are major stationary phase-induced proteins [57].

The use of hot water or steam may be a possibility to replace disinfection. Martín-Diana et al. [58] reported that short time exposure of fresh-cut (FC) lettuce to water steam reduced the respiration rate (RR), partially inactivated browning-related enzymes, and kept the mesophilic load as low as with a chlorine treatment. In FC fruits, mild heat pre-treatments (MHPT) (40 °C/70 min or 46 °C for 75 min) were effective in inducing firmness and avoiding browning of the cut surface while preserving their nutritional quality [59,60]. In the post-harvesting of fresh-cut vegetables hot water immersion treatment (HWT) and hot water rinsing and brushing (HWRB) technologies were successfully used [61]. HWT is applied at temperatures between 43 °C and 53 °C for periods of several minutes for the fresh cut, while HWRB is employed commercially for 10-25 s at temperatures between 48 °C and 63 °C. Additionally, oscillating magnetic fields (ohmic heating, dielectric heating, microwaves) represent alternative ways to heating food matrices. The application of high temperatures has been widely used for the elimination of foodborne pathogens. This is due to the effectiveness of heat and its ability to cause damage to diverse structures and components in microbial cells including outer and cytoplasmic membranes, RNA and DNA. It also causes protein denaturation leading to destruction of enzyme activity and enzyme-controlled metabolism in microorganisms [62,63]. Traditional heat treatment techniques widely adopted in food industry, such as pasteurization and sterilization are too harsh to be resisted by vegetative bacterial cells. An interesting concept has been also proposed on sensing environmental stresses including heat. According to this theory, cells produce extracellular proteins to sense stress and act as “alarmones” of the emergent environmental changes [64]. The production of these extracellular components are suggested to provide early warning of stress compared to the cytoplasmic membrane and ribosomes. Following stress-sensing, cells set up strategies to cope with the emergent hardship. These mechanisms involve changes of gene expression and protein activities aiming at preventing or reducing damage to cellular structures and components. An important change is the induction of the synthesis of the so-called “heat shock proteins” (HSPs) [65]. These are highly conserved proteins that act as molecular chaperones or proteases affecting protein folding, repair and degradation under normal and stress conditions [66]. For example, during heat stress, several HSPs such as DnaJ, DnaK, GrpE and GroEL function as chaperones preventing or repairing protein misfolding and thus ensuring their proper functioning. Whereas, other HSPs including ClpP, ClpX and Lon act as proteases catalyzing the degradation of misfolded proteins generated by exposure to stress. Both functions of HSPs help to provide cells with functional proteins which allow survival or growth during heat stress. In a study on heat shock proteins synthesis and heat resistance of Salmonella typhimurium, Mackey and Derrick [67] found four important heat shock proteins, with molecular weights ranging from 25 to 83 KDa, and correlated them with DnaK and GroEL reported in E. coli. The chaperone GroEL was found to diminish heat inactivation of a range of enzymes in vitro [68].

A decontamination step with lactic acid was evaluated to reduce the microbial contamination of minimally processed vegetables [69]. The antimicrobial effect of 1% and 2% lactic acid was established in potable tap water. Citric acid has been widely accepted as effective in reducing superficial pH of cut fruits [70]. Well documented is the antimicrobial effect of the treatments based on calcium salts on fruits and vegetables [58]. While calcium has a prevalent technological importance, on the other hand, organic calcium salts, with lowering pH (i.e. calcium lactate), may have antimicrobial properties [71].

In recent years, the acid stress response of several prokaryotes has been studied with both proteomics and transcriptomics approaches. A few reports describe the use of these approaches to study the organic acid stress response caused by lactate [73], acetate [74], propionate [74] and formate [75]. In other prokaryotes some enzymatic-transport systems have been found to play a role in pH control or in the maintenance of the proton motive force: a proton-translocating F₁ F₀ -ATPase [76]; several sodium-proton antiporters [77]; amino acid decarboxylases that use an intracellular hydrogen ion for the decarboxylation of an imported amino acid [76,78]. Several theories have been postulated to explain the toxic effect of organic acids in more detail. One of these considers organic acids as uncouplers that transport protons towards the inside of the cell, which is a pH-driven process. Eventually, this influx could lead to a complete dissipation of the proton motive force [79]. A second aspect relates to the deleterious effects of the lower intracellular pH, caused by the lactic acid. However, whereas many organisms aim at maintaining a constant intracellular pH [80], most anaerobic fermenting species avoid a basic pH keeping a lower intracellular pH, and, as a result, increasing their tolerance to organic acids [81]. A third factor explaining the inhibitory effect of organic acids is the intracellular accumulation of anions, which could lead to both end-product inhibition and a loss of water activity (a_w) [82].

A link between acid, ethanol and stress response has been demonstrated in a number of Gram-positive bacteria including food-borne pathogens. The expression of some stress inducible genes identified so far in bacteria is also affected by low pH values and ethanol, suggesting the intersection of different regulatory pathways and overlapping control of gene expression. For instance, acid and solvents such as ethanol and buthanol, induces several heat shock proteins, including DnaK and GroEL. Moreover, the Clp-ATP dependent proteases which degrade aberrant and nonfunctional proteins, arising from stress conditions are also responsible for adaptation to multiple stresses and are inducible by low pH or high ethanol [83].

Ethanol kills organisms by denaturing their proteins and dissolving their lipids and is effective against most bacteria, but is ineffective against bacterial spores [84]. Additionally, ethanol causes water stress by lowering a_w and thereby interferes with hydrogen bonds within and between hydrated cell components, ultimately disrupting enzyme and membrane structure and function [85].

Plotto et al. [86] tested ethanol vapours on fresh cut fruit and showed that at lower application rates (8–10 h exposure), ethanol could be used as a safe microbial control in a fresh-cut production sanitation system. Ethanol vapors applied to whole apples reduced ethylene and CO₂ production of fresh-cut apples, and their shelf life was increased due to maintenance of visual quality [87].

Chlorine dioxide is a stable dissolved gas, having a high oxidation and penetration power; ClO₂ is a strong bactericide: with minimal contact time, it is highly efficient against pathogenic organisms such as Legionella, Amoebal cysts, Giardia cysts, E. coli, and Cryptosporidium [88]. Hyrdogen peroxide is a powerful bactericide (including spores) and oxidant, being able to generate other cytotoxic oxidising chemical species such as hydroxyl radicals [89]. Electrolyzed water (EW) is formed by adding a very small amount of NaCl (usually about 0.1%) to pure water, and conducting a current across an anode and cathode [90], the cathode area produces alkaline reducing water while the anode area produces acidic oxidizing water [90]; upon release of O•, O₃ acts as a strong oxidizing agent being very effective in destroying microorganisms [91]. O₃ destroys microorganisms by the progressive oxidation of vital cell components, preventing microbial growth and extending the shelf-life of many fruit and vegetables, and its industrial use is increasing [92].

Oxidative stress is a key stress in bacteria, caused by an imbalance between intracellular oxidant concentration, cellular antioxidant protection and oxidative change of macromolecules (membrane lipids, proteins and DNA repair enzymes) [93,94]. The reactive oxygen species (ROS) and nitrogen species (RNS) are the main causes of oxidative stress [95]. They are principally constituted by the hydroxyl radical (^•OH), the superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), organic hydroperoxide (ROOH), peroxynitrite (OONO) and nitric oxide (NO). ROS and RNS cause damages to proteins [96], DNA molecules [97], RNA and lipids leading to negative repercussions of the cellular metabolism functions [98]. The toxicity of ROS/RNS discloses the significant role of competent protection subsystems, for instance the detoxification subsystem that numbers enzymes classified with regard to their substrates, or thioredoxin that help in the cellular defense against several oxidative stresses [99]. Catalases are common enzymes found in almost all-living organisms that catalyze the decomposition of hydrogen peroxide to produce oxygen and water [100]. Peroxidases reduce hydrogen or organic peroxides into water and alcohol moiety. This class of enzymes includes a wide number of phylogenetically unrelated families such as peroxiredoxins [101], rubrerythrins [102], glutathione-peroxidases [103] or haloperoxidases [104]. Superoxide dismutases (SOD) dismute superoxide into hydrogen peroxide and oxygen [105]. An additional mechanism lately reported involves superoxide reductases (SOR), that are non-heme iron proteins [106]. The latter catalyzes the one-electron reduction of superoxide into hydrogen peroxide. Moreover, RNS-scavenging enzymes are essentially globins and nitric oxide reductases [107].

The inner osmotic pressure of a bacterial cell is normally maintained higher than that of the surrounding medium [108,109], and this is generally referred to as “turgor pressure”. To maintain this turgor pressure in a medium with a high concentration of solutes and to mitigate the osmotic stress resulting from low water availability, microbial cells tend to increase their internal cytoplasmic solute concentration through different mechanisms [110]. Water availability in a food environment is usually assessed by measuring the water activity (a_w). Water activity (a_w) is defined as the ratio of the water vapour pressure of the food or solution to that of pure water at the same temperature. It indicates the quantity of water available in a material for microbial growth. The values of water activity are represented in a scale range between 0 (no available water) and 1 (pure water). While most bacteria show rapid growth at high a_w (0.99), microbial growth does not occur at a_w below 0.6 [111]. Several osmosensors are known to be involved in osmotic stress induced responses [112]. As mentioned above, the solute concentration in the bacterial cytoplasm is normally maintained above that of the external environment. However, immediately, following an osmotic up-shift (decrease in a_w) in the environment, bacterial cells respond by activation of transporters that aid the cell increase the internal solute concentration by either uptake of inorganic ions into the cell or synthesis and concentration of specific organic solutes to counter the osmotic stress [113]. Under mild osmotic stress, only the ionic solutes are accumulated, whereas other compatible solutes become progressively more important on exposure to severe osmotic stress [114]. These accumulated solutes must not interfere with biochemical processes within the cell and they are thus termed “compatible” solutes [108]. Potassium ions (K⁺), glutamate (as ionic solutes), glycine betaine, trehalose and proline (as non ionic solutes) are the most important compatible solutes accumulated by bacterial cells [114,115]. Members of the Enterobactereaceae family are reported to synthesize glutamate and trehalose, while K⁺, glycine betaine and proline are taken up from the medium [110]. Accumulation of K⁺ during osmotic stress takes place in the initial response, which is then accompanied by increased synthesis of glutamate to preserve electroneutrality in the cytoplasm. This is followed by the accumulation of other compatible solutes such as glycine betaine, proline and trehalose [114,115]. The accumulation of the latter molecules was found to influence that of the originally accumulated compatible solutes since increased levels of trehalose were associated with decreased accumulation of K⁺ and glutamate [114]. The above mechanism of solute accumulation appears to be affected by environmental temperatures as trehalose accumulation was reported to be enhanced at higher temperatures (45°C) in S. typhimurium [116].

Radiations have been used both to delay ripening-associated processes and to diminish microorganism growth. Several studies have been published, and in recent times, UV-C has been used as an alternative treatment to preserve the quality of different fruits and vegetables [117]. The use of non-ionizing, germicidal and artificial UV at a wavelength of 190–280nm (UV-C) was found to be effective for surface decontamination of fresh-cut products. Lado and Yousef [118] reported that UV-C radiation from 0.5 to 20 kJm⁻² inhibited microbial growth by inducing the formation of pyrimidine dimers which alter the DNA helix and block microbial cell replication. Therefore, cells which are unable to repair radiation damaged DNA die and sub-lethally injured cells are often subject to mutations. Treatment with ultraviolet light is simple to use and lethal to most types of microorganisms [119]. Intense light pulses (ILP) are an interesting decontamination method for food surfaces approved by the US Food and Drug Administration (FDA) that could be appropriate for disinfecting fresh-cut produce. ILP kills microorganisms using short time (from 85 ns to 0.3 ms) high frequency pulses (from 0.45 to 15 Hz) and energy per pulse ranging from 3 to 551 J of an intense broad spectrum, rich in UV-C light [120]. This treatment seems to induce structural changes of microbial DNA, similarly to the effect caused by continuous UV sources, although further mechanisms seem to be involved [121].

Irradiation of DNA with UV light produces a variety of photoproducts, of which the main species are cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone. Both lesions, if not repaired, provoke mutagenesis and cell death. To survive in a UV-rich environment, E. coli developed an inducible response known as the SOS response regulated by the recA-lexA regulon. The SOS response aids survival by combining increased expression of genes involved in Nucleotide Excision Repair (NER) and recombinational repair mechanisms. The genes recA for the recombination enzyme, RecA, and uvrA and uvrB for subunits of the UvrABC NER enzymes, UvrA and UvrB, have SOS boxes that are bound by the LexA repressor under physiological conditions. Upon UV irradiation, the constitutive amount of RecA protein binds single-stranded DNA resulting from replication blocks and acts as a coprotease for inactivation of LexA, consequently the levels of RecA, UvrA and UvrB increase. Upon completion of repair, the inducing signal disappears and cells return to the preinduction state [122].

High pressure treatments of fruits and vegetables have been applied on processed products typically having been processed to some degree [123,124]. High pressures have been used to inhibit enzymes, microorganisms and spores, and to preserve aroma compounds [125]. Yanga et al. [126] indicate an undesirable effect of hyperbaric storage on the synthesis of peach volatiles immediately after storage, however, the post-storage potential for recovery of normal synthesis has not been assessed.

Pressure effects on any physiological or biochemical system basically result from the compression of the system, according to Le Chatelier’s principle, which states that at equilibrium a system tends to minimise the effects of troubling external factors. In lipid membranes, a pressure increase of 1,000 atm is equivalent to a temperature decrease of 20 °C [127]. Pressure increase and temperature decrease result in similar effects, i.e. by ordering structures and reducing flexibility in lipids, nucleic acids and carbohydrates [128]. For proteins, pressure and temperature act in synergy and promote protein denaturation and loss of function [129]. In E.coli O 157:H7, high pressure affected the transcription of many genes involved in a variety of intracellular mechanisms, including the stress response, the thioldisulfide redox system and the Fe-S cluster assembly [130].

Producers mainly rely on produce sanitation, refrigeration temperatures, and, more recently MAP to extend shelf life and to reduce microbial load [131]. Modifying the internal atmosphere of a package lowers the oxygen (O₂) concentration, from 20% to 0%, hence slowing down the growth of aerobic organisms and the speed of oxidation reactions. The removed oxygen can be replaced with nitrogen (N₂), commonly acknowledged as an inert gas, or carbon dioxide (CO₂), which can inhibit the growth of bacteria. Although there is a wide literature describing how MAP affects microbial load on various food-produce [132,133], little is known about how growth under subatmospheric oxygen partial pressures would impact the enteric pathogens’ ability to breach the gastric stomach barrier and increase the risk of disease. CO₂ inhibits the growth of bacteria by (i) affecting cellular enzymes and decreasing the rate of metabolic reactions, (ii) CO₂ product repression of carboxylases and decarboxylases, (iii) disrupting cell membrane structural integrity and/or specific functions, (iv) decreasing the substrate and intra-cellular pH, or by a combination of these mechanisms [134]. The extent of inhibition by CO₂ varies with the microorganism, CO₂ concentration, temperature of incubation, and type of food [134,135].

Greater consumer awareness and concern regarding synthetic chemical additives have led researchers and food processors to look for natural food additives with a broad spectrum of antimicrobial activity [136]. This is an heterogeneous category: some examples. Plant essential oils and natural aroma compounds are gaining interest for their potential as preservative ingredients or decontaminating treatments, as they have GRAS status and a wide acceptance from consumers [137,138]. The antimicrobial components are commonly found in the essential oil fractions and it is well established that many have a wide spectrum of antimicrobial activity, with potential for control of L. monocytogenes and spoilage bacteria within food systems [139]. Oregano (Origanum vulgare) and thyme (Thymus vulgaris) are amongst the most active EOs, while lemon balm (Melissa officinalis) and marjoram (Origanum majorana) exhibit a good antimicrobial action against Gram-positive and Gram-negative bacteria, respectively [140]. Chitosan, which is a cationic polysaccharide extracted from source of shellfish exoskeletons or the cell walls of some microorganisms and fungi, has been used to preserve the quality of post-harvest fruits and vegetables [141,142]. Martin-Diana et al. [143] tested whey permeate at different concentrations (0.5%, 1.5% and 3%) in the washing treatment of lettuce and carrots, and the results suggest that whey permeate could be a promising alternative for sanitizing fresh-cut vegetables.

Packaged produce harbour large populations of microorganisms including pseudomonads, lactic and bacteria (LAB) and Enterobacteriaceae [28,43]. The background microflora provide indicators of temperature abuse largely by causing detectable spoilage, and levels can vary appreciably for each product and during storage. LAB can exert antibacterial effects due to one or more of the following mechanisms: lowering the pH; generating H₂O₂; competing for nutrients; and by producing antimicrobial compounds, such as bacteriocins [45]. Cai et al. [144] reported that a large portion of LAB isolates from beansprouts inhibited the growth of L. monocytogenes. Strains of LAB were reported to inhibit Aeromonas hydrophila, L. monocytogenes and S. typhimurium, on vegetable salads [145]. Various researchers have reported antagonism by the native microflora of vegetables against Listeria [146,147]. Reducing the background microflora of endive leaves and shredded lettuce resulted in enhanced growth of Listeria [148]. However, the inhibitory effects were dependent on gas atmosphere; in 3% O₂ (balance N₂) growth of the mixed population was inhibited while L. monocytogenes proliferated [149]. Enterobacter isolates significantly reduced L. monocytogenes growth during storage on a model lettuce medium; however, the inhibitory activities of Enterobacter decreased as the concentration of CO₂ increased [149]. Competitive microflora had a significant effect on the growth of E. coli O157:H7 in broth media [150]. Little is known about the mechanism by which Salmonella manages to compete with natural microflora and survive on plant products [151]. Generally, the complex interactions with the indigenous microflora may have significant effects on survival, growth and biocontrol of pathogens.

Bacteriocins are antimicrobial peptides or proteins produced by strains of different bacterial species. The antimicrobial activity of this set of natural substances against foodborne pathogenic, as well as spoilage bacteria, has raised considerable interest for their application in food preservation [152].

Nisin is the only commercially available bacteriocin recognized as a safe and legal biological food preservative (number E234) by the Food and Agriculture Organization and World Health Organization as well as the FDA. Nisin, a broad-spectrum, pore-forming bacteriocin, is produced by lactic acid bacteria that are often found on food-produce [153]. It is active against many Gram-positive bacteria, including L. monocytogenes [154]. Nisin is particularly active at the lower pH values typical of many fruits and some vegetables [155].

The production of bacteriocin must thus be coupled with a mechanism by which the producing strain can protect itself from the lethal action of its own antimicrobial compound. This mechanism is referred to as immunity. In non-nisin-producing Lactococcus lactis, nisin resistance could be conferred by a specific nisin resistance gene (nsr), which encodes a 35-kDa nisin resistance protein (NSR). NSR is a nisin-degrading protease [156], however, the mechanism underlying NSR-mediated nisin resistance is poorly understood.

Leverentz et al. [157] reported a study on the control of Salmonella by phages on fresh-cut fruits. The use of naturally occurring lytic phages to reduce contamination of fresh-cut produce with foodborne pathogens has several advantages over the use of chemical sanitizers and washes [38]. For example, methods commonly used in industry, such as aqueous washes containing chlorine formulations or plain water, are nonspecific and can achieve a less-than-10-fold reduction in Listeria populations on cut-produce surfaces [158]. Conversely, specific phages attack the targeted pathogens only, thus preserving the competitive potential of the indigenous microflora [38]. Leverentz et al. [159] found that treatment with a Listeria-specific lytic phage cocktail alone or in combination with nisin is an effective method for reducing L. monocytogenes contamination on fresh-cut fruit.

Bacteria have evolved different sophisticated natural bacteriophage defense systems that can interfere with bacteriophage proliferation at different steps during the lytic cycle. These consist of natural phage defense mechanisms that impede the adsorption of the bacteriophage to the cell, mechanisms that inhibit the injection of DNA into the cell, restriction-modification systems, and numerous systems that abort the infection at various points in the replication cycle [160,161,162]. Additionally, Hazan and Engelberg-Kulka [163] demonstrated that E. coli mazEF-mediated cell death acts as a suicide-defense mechanism to protect the bacterial culture against the spread of P1 phage infection.