mini review: antimicrobial strategies in the production of fresh-cut

Mini review: Antimicrobial strategies in the production of fresh-cut lettuce products

Ö. Tirpanalan , M. Zunabovic, K. J. Domig and W.Kneifel

Department of Food Science and Technology, BOKU - University of Natural Resources and Life Sciences, Vienna

As the consumer attitudes shift to healthier, low caloric value as well as to convenience products, the demand for whole and fresh-cut produce has proportionally increased over the last decade [19, 53, 55]. Together with the grown market, an increased number of outbreaks associated with fresh-cut produce brought the necessity to deal with microbial decontamination methods of the fresh-cut lettuce products. Between 1994 and 2011, more than twenty alerts have been notified through the RASFF portal of the EU regarding hazardous pathogenic microorganisms associated with fresh lettuce. Most frequently involved pathogens are Salmonella serotypes followed by Campylobacter spp. and Norovirus genotypes. Additionally, Listeria monocytogenes, E. coliO157:H7 and Shigellasonnei were reported as sources of outbreaks. The microbial load of naturally occurring spoilage or even pathogenic microorganisms associated with these products may belong to a broad diversity depending on the production hygiene, storage conditions and on the fact whether whole or fresh-cut vegetables are considered [1].

The fresh-cut produce manufacture has recognized the application of chlorine solutions as one of the most common techniques for the treatment of vegetables [20, 54]. However, health- and environment-related concerns regarding the carcinogenic by-products of chlorine have promoted the search for alternative methods to decontaminate fresh-cut products [48, 54]. Nevertheless, difficulties have been observed to develop sufficiently standardized applications of chemicals as sanitizing agents for the washing of vegetables, as different concentrations of chemical sanitizers were suggested. Hence, the use of chlorine for food disinfection is generally tolerated without being authorized [8]. In parallel, the application time and the temperature of the disinfectant and its combinations with other chemicals or physical methods may exert various effects on microbial reduction of different spoilage and pathogenic microorganisms [7, 20]. As an alternative, chlorine dioxide has been applied. The advantage of this compound is not to form chloramines that are reactive with organic matter [50, 65]. Hydrogen peroxide, ozone and organic acids are still not commonly applied in the industry in this context. However, there are several scientific studies illustrating their efficacy and applicability [7, 20, 50, 54]. Additionally, the scientific community is wondering if antimicrobials naturally occurring in plants or of animal and microbial origins are useful as decontamination agents applied to vegetables through washing procedures. A totally different branch of decontamination treatment is the use of irradiation technologies [51]. Only recently, the Scientific Committee on Food of the European Food Safety Authority (EFSA) has published an updated scientific opinion regarding safety concerns related to irradiated food (EFSA, 2011). UV treatment is also suggested as a useful technique for reducing the microbial load of the product. Last but not least, the application of electrolyzed water that forms free oxidants without adding chemicals other than sodium chloride has been considered [48].

In conclusion, there is some growing need for having a survey of scientific data and practical experiences related to the various decontamination techniques, their efficacy and usefulness in being applied to control the microbial load of fresh-cut produce. Therefore this chapter will evaluate the available knowledge also providing some information on their advantages and disadvantages in industrial application.

Keywords: fresh-cut lettuce; decontamination; chlorine solution; irradiation; E. coli O157:H7; Listeria monocytogenes;

Introduction

Consumption of fresh produces has gained demand over the last decade [8]. This is mainly due to the fact that, today´s consumers are more aware of the nutritional and health benefits of fresh produces. As the consumption rate of fresh vegetables increases, the space devoted in supermarkets to these products increases proportionally [48]. Especially in recent years the market of minimally processed vegetable (MPV) has grown rapidly due to its convenience degree. Together with the reduced time for preparation, lower transportation and less storage cost make MPV favorable, not only for the home consumption but also for the gastronomy. However, the pH of the lettuce (5.5-6.0) together with the high aw-value and cut/broken surfaces/tissues serve ideal conditions for the microbial growth that reduces the shelf life. MPV industry aims to serve the consumer an extended shelf-life product together with the ensured safety and maintained nutritional and sensorial values. As a definition, minimal processing does not cover intense preservation steps on the production line, such as heat sterilization, freezing or drying, thus, physiological activities of food, like respiration, continue throughout the shelf-life. Minimal processing for the fresh-cut iceberg lettuce includes the steps of harvesting, cold storage, trimming, shredding, washing/rinsing, draining, packaging, cold storage and finally distribution [8]. The storage temperature plays a critical role in preserving safety and quality of the product. The cold chain throughout the processing, distribution and storage should be kept at below 10 ◌۫C [8]. Indeed, the French regulation requires keeping the fresh-packed products at 0-4 ◌۫C and the U.K. Food Hygiene Regulation directs it as

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below 8 ◌۫C [17], while the German Society for Hygiene and Microbiology (DGHM) advises to keep the product below 6 ◌۫C emphasizing that 6 days should not be exceeded the use-by-date. The main decontamination step of MPV is washing. Conventionally processed fresh-cut lettuce has the washing step after shredding/slicing/cutting, generally by dropping into a washing tank containing sanitizer [8]. The application parameters such as time, temperature, pH vary regarding the antimicrobial added to water. Among the sanitizers added to washing water the most common one in the industry is chlorine. Chlorine is added generally in the amount of 50-200 ppm as free chlorine [8,18, 32]. Nevertheless, in some European countries like Germany, Switzerland, Denmark and Belgium, the application of chlorine for the production of food as wash water disinfectant is restricted [32, 48, 54]. Thus, washing with warm water is also an accepted method on the decontamination of MPV. The United States Code of Federal Regulation suggests the ozone in the processing of food as a safe antimicrobial agent. Furthermore, chlorine dioxide is also accepted by the Food and Drug Administration (FDA) for the use in washing vegetables. In spite of the high bactericidal and sporicidal activity of hydrogen peroxide, it is not preferred by the fresh-cut industry because of its negative effect to severely browning of the lettuce leaves [54]. Recently, organic acids especially ascorbic acid, citric acid and lactic acid are tempted to be utilized as the decontamination agent for vegetables. The combination of chlorine with organic acids has promoted the efficacy compared with either one of them. The application of organic acid decreases the pH of the chlorine solution making it more effective but also more corrosive to the food equipment and less stable [69]. Among the physical methods it is reported that acidic electrolyzed water has a similar activity with chlorine and a greater effect than ozone on lettuce [36]. Similarly, application of gamma ray irradiation which is another physical method, promises to extend the shelf life of MPV [7, 54]. The application of irradiation with a maximum level of 4.0 kilogray (kGy) on lettuce and spinach is approved by FDA. Nonetheless, the recently updated scientific opinion of EFSA permits the application of irradiation to vegetables with a maximum level of 1 kGy. Applied kGy doses to different produces play a critical role regarding the amount of log unit and the type of organisms that are reduced. Although 1 kGy is enough to eliminate the risk of pathogenic vegetative bacteria, viruses are more resistant to the treatment, having D10 values around 3 kGy [13].In the mentioned opinion of the scientific committee of EFSA, it is also pointed out that the dose above 0.5 kGy causes softening on lettuce [44]. Regarding the physical method through UV application there is only limited data clarifying the efficacy of the method to decontaminate the lettuce. More data are available regarding the UV application on smooth surface vegetables such as, potatoes, apples and tomatoes [4]. Available data show that the inhibition on the microbiological load depends on the dose of UV treatment. Short length UV light is more effective at higher doses, however, as the dose increases the respiration rate of the lettuce increases proportionally, resulting in a decrease in the sensory quality [4].

1. Chemical Sanitizers

1.1 Chlorine

The common form of chlorine that is used in the food industry is sodium hypochlorite (NaOCl). The outputs of the sodium hypochlorite-water reaction are hypochlorous acid (HOCl) and sodium hydroxide (NaOH) and the further dissociation of hypochlorous acid is the hypochlorite ion (OCl-). The term of free chlorine does not include combined chlorine compounds that are not available for the oxidative reactions such as chloramines [7]. Although hypochlorous acid (HOCl) is more effective than the hypochlorite ion (OCl-), under alkaline conditions which are promoted by the formation of NaOH the equilibrium shifts to the hypochlorite ion (OCl-) [32]. It is reported that at pH 7.0, which is the target pH for the fresh-cut industry, the percentages of HOCl and OClare 78% and 22% respectively [12]. Despite the fact that the concentration of HOCl increases as the pH decreases, the solution becomes corrosive to the food contact equipments at lower pH values [8, 32]. The applied commercial amount is between 50 and 200 ppm of free chlorine with a variable contact time from 1 to 10 min or even longer, at chilling temperatures [8, 18, 32]. Nevertheless, many studies have reported that the reduction of the microbiological load is accomplished mainly in the first minute and further reduction with increased contact time is not significant [32, 51]. There are several parameters influencing the efficacy of hypochlorite based washing systems such as the application temperature, the pH of the solution, washing time and the amount of organic matter present. Chlorine can bind organic material and form bound chlorine which is less effective than the free chlorine as biocide. If the amount of the organic material in the produce is increasing the efficacy of the washing system is reduced [32]. Therefore the inconsistency of reduction levels of strains belonging to the same species with the same application amount might result from the surface area of cut lettuce pieces, due to the processing, altering the amount of free chlorine in the solution. Apart from that, the initial microbiological loads in the lettuce as well as the applied temperature are also parameters affecting its efficacy. Moreover, the experiments, in which the microorganisms are cultured and inoculated in the laboratory environment, demonstrate greater efficacy than those investigating the reduction of naturally present microorganisms. The reason of easier inactivation of artificially inoculated cell cultures has been suggested as stronger cell attachment of the natural microbiota [34].

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The biggest concern of chlorine based systems is the formation of by-products like haloketons, haloacetic acids, trihalomethans, and their effect on the environment and on human health [20]. These by-products are recognized as carcinogenic, although negative health effects on human are not investigated and described in detail. For that reason the use of chlorine for the ready-to-use food products is prohibited in some European countries like Germany, Switzerland, the Netherlands, Denmark and Belgium [32, 48, 54]. Nonetheless, in many other European countries there is a lack of regulatory issues specifying the application technique of chlorine for food. Furthermore, it has been reported that there is a lack of knowledge in the industry how to fully optimize these systems regarding the pH and free chlorine amount which is affected by the organic matter present [58]. On the contrary, the powerful oxidizing effect of chlorine, being soluble in water, easy to apply and its relative low cost make it one of the most widely used sanitizers among the U.S. and some European countries, to decontaminate the fresh produce [7, 8, 32, 54].

Table 1: Relative efficacy of chlorine (Cl2) wash applications for the decontamination of lettuce

Type of microorganism

Amount of free chlorine (ppm)

Application time (min)

Log10cfu/g reduction

Reference

Yersinia enterocolitica

100 10 ~ 2 [14] 300 ~ 3

Total aerobic count 200 5 1.3 [62] Salmonella

100 10 0.7 [7] 1 0.9 [32]

5 1.1 Salmonella Typhimurium DT104

200 10 ~ 1 [35]

L. monocytogenes (NTCT 7973,NCTC 5214, ATCC 19116, LM 206, LM 168)

100 1

0.7 [26]

L. monocytogenes

1.3 [32] 5 1.8

L. monocytogenes(81-861, Scott A, 537, 540, 845)

200 10 1.7 [69]

E. coli O157:H7 200 10 1.2 [35] E. coli O157:H7 (C9490, ATCC 35150)

300 3 ~ 0.5 [45] 600 3 ~ 0.5

E. coli ATCC 25922 100 2 2.5 [50] Staphylococcus aureus FDA 209P

200 10 1.4 [35]

1.2 Chlorine dioxide

Chlorine dioxide (ClO2), as a sanitizing agent causes reduction in microbial population by disrupting the protein synthesis and membrane permeability of the bacterial cell [9]. It has been used since 1944 for the treatment of water and in 1998 aqueous chlorine dioxide was permitted by FDA for the use as sanitizing agent for fruits and vegetables [59]. In the Code of Federal Regulation document (FDA, title 21, part 173.300) it is stated that the amount should not exceed 3 ppm as residual chlorine dioxide, additionally it is directed that the treatment with chlorine dioxide should be followed by rinsing with potable water. The information about the efficacy of aqueous chlorine dioxide is quite limited compared to the hypochlorite solutions. As in the case of HOCl solution, for the chlorine dioxide the reduction on microbiological load also depends on the application parameters (See table 2). Chlorine dioxide has several advantages over chlorine solutions; one of those is not being affected by pH. Additionally, it has reduced cross-reactivity with organic materials [32, 51, 54, 59]. It does not form chloramines by reacting with ammonia and compared to chlorine fewer organohalogens are formed as reaction products of ClO2 [9]. Furthermore, it has 2.5 times greater oxidizing power compared to chlorine. However, chlorine dioxide is unstable; therefore it must be generated on site, additionally, it can be explosive at high concentrations and it decomposes when it is exposed to light at a temperature higher than 30 ◌۫C [9, 32]. Another disadvantage observed for chlorine dioxide is the fact that the application of 5 mg/L gaseous chlorine dioxide for 14.5 min caused deterioration on visual quality [7]. Adverse changes have also been reported in the sensory quality of lettuce on the 3rd day of storage after the treatment with 1.4 mg/L chlorine dioxide for 10.5 min [60].

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Table 2: Relative Efficacy of Chlorine dioxide (ClO2) wash applications for the decontamination of lettuce


Amount of ClO2(ppm)



Reference

L. monocytogenes (81-861,Scott A, 537, 540, 845)

5 10 1.1 [69] 3 10 0.4

3 5 0.4 E. coli O157:H7 (C7927, EDL933, 204P)

5 10 1.2 [59] 5 5 0.98 10 5 1.3 10 10 1.7 20 5 1.4 20 10 1.7

Total aerobic count 1.5 (gaseous) 5 2.5 [62]

1.3 Hydrogen peroxide

Hydrogen peroxide (H2O2) has the ability of generating cytotoxic oxidizing agents like hydroxyl radicals which oxidize the cell membrane, biomolecules and DNA of microorganisms, therefore it possesses bactericidal and sporicial activities [51]. Hydrogen peroxide has been used as sterilizing agent for food contact surfaces, packaging material and aseptic filling. It is classified as generally recognized as safe (GRAS) and it is allowed for the use of water and surface disinfection. Several studies show that hydrogen peroxide is effective to reduce the microbiological load on some fresh cut produces such as, alfalfa sprouts, bell peppers, cucumber, cantaloupe, without altering the sensory quality [9, 52]. Nevertheless, the dipping treatment in the solution of H2O2 resulted in severely browning to shredded lettuce [51]. Thus, it is not preferred to decontaminate fresh-cut lettuce. The main advantage of hydrogen peroxide is that it is degraded into water and oxygen, therefore does not leave critical residues, however the degree of degradation, accordingly amount of residues, depend on the amount of catalase enzyme (peroxidase) available in the produce item [51, 57]. Alternatively to H2O2 , the microbiological efficacy of peroxyacetic acid, (the equilibrium mixture of acetic acid and hydrogen peroxide) to decontaminate the lettuce, has been studied [26, 47, 55]. Furthermore, it is suggested that peroxyacetic acid is more effective than chlorine with similar contact times at killing some pathogens [7]. The effect of aerosolized peroxyacetic acid on L. monocytogenes, SalmonellaTyphimurium and E. coli O157:H7 has been examined. (See table 3) [47]. Applications demonstrated drastically increased log reduction values as the time increased from 10 to 30 min. In the same experiment, a 60 min treatment resulted in even higher reduction values. However, application of the agent for longer residence time is not appropriate and realistic for the industry.

Table 3: Relative efficacy of peroxyacetic acid wash applications for the decontamination of shredded lettuce


Amount of H2O2(mg/L)



Reference

L. monocytogenes (ATCC 19113, ATCC 19114, ATCC 7644)

40 (aerosolized)

10 2.5 [47] 30 2.7

L. monocytogenes (NTCT 7973, NCTC 5214, ATCC 19116, LM 206, LM 168)

50 1 1.7 [26]

L. monocytogenes (CWD 95,CWD 201, CWD 249)

80 5 4.6 [55]

E. coli O157:H7 (ATCC 35150, ATCC 43889, ATCC43890)

40 (aerosolized)

10 0.8 [47] 30 2.2

E. coli O157:H7 (AR,AD305,AD317)

80 5 4.3 [55]

Salmonella Typhimurium (ATCC 19858, ATCC 43174, ATCC 363755)

40 (aerosolized)

10 0.3 [47] 30 3.3

Total aerobic count 80 5 ~ 1 [62] 250 2.4

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1.4 Ozone

Ozone (O3), which is naturally occurring as triatomic oxygen molecule, has a strong oxidation-reduction potential that can inactivate the contaminants by reacting directly as molecular ozone or through the derived free radicals [32, 50]. Ozone has been used for water treatment for many years and in 1997 the U.S. expert panel classified ozone as Generally Recognized as Safe (GRAS) for the use in food processing [21]. Its antimicrobial action results from the cell membrane oxidation, also it has about 50% greater oxidation capacity than chlorine. Ozone decomposes into oxygen, not forming any by-products [32, 34]. In the presence of organic matter it forms aldehydes, ketones, carboxylic acid causing less regulatory concerns [24]. There are several studies investigating the different application methods (bubbling ozone, ozonation with low and high speed stirring, gaseous ozone, aqueous ozone, ozonation combined with sonication) to maximize the decontamination efficacy of ozone (See table 4). Among the different application methods bubbling ozone has been found the most effective one [34, 50]. As the temperature decreases the solubility of the ozone in water is increasing. Therefore ozone is more effective on inactivating the microorganisms at lower temperatures [50]. It is reported that the log reduction of artificiallyinoculated E. coli indicates a difference depending on the incubation time [50]. For instance, E. coli incubated for 6 hours after the treatment with bubbling ozone show a 2.54 log reduction while incubated for 18 hours E. coli show a 2.18 log reduction after the same treatment. The reason of this modest decrease can be explained by increased attachment of cells to lettuce surface over time. This also contributes to the suggestion that the combination of ozone treatment with mechanical force such as bubbling, stirring, gives higher log reductions due to the loosening of the cell attachment [34]. It is reported that a concentration of 3 ppm for 5 min reduced the L. monocytogenes and E. coli O157:H7 counts below the detection limit when it is applied to the whole lettuce with initial counts of 5.9 log cfu/g as well as to the shredded lettuce with initial counts of 6.0 log cfu/g L. monocytogenes and E.coliO157:H7 in different batches. The same application resulted in a 4 and a 1.6 log cfu/g reduction to the mesophilic bacteria and yeast counts respectively (See table 4). Although molds were below the detection limit, on the 5th day of storage at 4 °C counts reached the initial load [55]. The main disadvantage of ozone treatment in food processing is the safety of employees. Possible health risks may result from intense exposure to ozone. Therefore, adequate ventilation systems are necessary to prevent accidental inhalation of ozone [50]. The possibility of discoloration and deterioration of the product flavor is pointed out [51]. Additionally, appropriate system adaption generating ozone results in high initial capital cost [54].

Table 4: Relative efficacy of ozone (O3) wash applications for the decontamination of lettuce products


Application strategy

Amount of O3

Application time [min]

Fresh produce type


Reference

Natural contaminating microbiota

Bubbling ozone

49 mg/L- 0.5 L/min

5 Shredded lettuce

1.9 [34]

Total aerobic count Ozonated water

5 ppm 10 Whole lettuce

1.5 [36]

E. coli

Ozonated water

1.5 ppm 2 Lettuce leaves

1.2 [50]

Bubbling ozone

30 g/h 2 Lettuce leaves

2.0

E. coli O157:H7

Ozonated water

5 ppm 5 Shredded lettuce

1.1 [68]

E. coli O157:H7 (AR, AD305, AD317)

Ozone by spraying

3 ppm 5 Whole and shredded lettuce

Not detectable

[55]

E. coli O157:H7

Ozonated water

9.7 ppm 10 Shredded lettuce

1.4 [59] 16.5 ppm 1.4

Gaseous Ozone

5.2 ppm 1.1 15 1.4

7.6 ppm 10 1.1

15 1.8 L. monocytogenes (CWD95,CWD201, CWD 249)

Ozonated water by spraying

3ppm 5 min Whole& Shredded lettuce

Not detectable

[55]

Total aerobic count Shredded lettuce

4 Yeast count (natural) 1.6 Mold count (natural) 2.4

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2. Natural Antimicrobials

2.1 Organic acids

It is well known that many pathogenic and spoilage microorganisms cannot grow at low pH values. Organic acids have antimicrobial properties by decreasing the pH of the applied solution. The dissociation of hydrogen ions causes reduction in the internal cellular pH of the organism. Disruption in the ability of the cell maintaining the pH homeostasis results in disruption of membrane permeability and substrate transport [32]. Organic acids (e.g. citric acid, tartaric acid, malic acid, sorbic acid, lactic acid, acetic acid) are known as weak acids having different inhibitory effects compared to strong acids. They acidify the cells´s interior by being lipophilic and penetrating the plasma membrane [3, 56]. The decline in intracellular pH results in inhibition of glycolysis and cell transport system [56]. Most of the organic acids are approved as Generally Recognized as Safe (GRAS) for food treatment, although their application percentages vary depending on the type of food. There are some studies investigating the efficacy of organic acids for the vegetable treatment. It is reported that 10 min of 50 mMfumaric acid treatment at room temperature is more effective than 200 ppm of chlorine treatment at the same temperature [35]. A comparable effect could be observed for 200 ppm of chlorine after 1 min treatment combined with a following heat treatment at 50°C. Nonetheless, the same authors pointed out that fumaric acid treatment promoted browning of the lettuce. It is suggested that dipping lettuce for 2 min into 0.5 % citric acid or lactic acid could be as effective as chlorine treatment [4]. However, by parallel investigation of different studies it might be suggested that the microbial reduction of the same species may vary with the specific strains probably because of the strain specific acid tolerance (see table 5).

Table 5: Relative efficacy of organic acid wash applications for the decontamination of lettuce products


Application method

Application amount [%]

Application time [min]

Fresh produce type


Reference

E. coli (ATCC 25922)

Dipping in lactic acid

0.5 2

Lettuce leaves (iceberg)

2.7 [4] 1 2.9

Dipping in citric acid

0.5 2.7 1 2.9 1 5 3.1

L. monocytogenes (ATCC 7644)


0.5 2

2 1

2.1

L. monocytogenes (3 strains)


1 Shredded lettuce (iceberg)

0.93 [68]

L. monocytogenes (81-861, Scott A, 537, 540, 845)


10 Shredded lettuce

~ 0.5 [69]

Dipping in acetic acid

~ 0.2

L. monocytogenes (ATCC 7644)


0.5 2 Lettuce leaves (iceberg)

1.4 [4] 1

1.6

L. monocytogenes (3 strains)


1

Shredded lettuce (iceberg)

1.0 [68]

Dipping acetic acid

0.6

E. coli O157:H7 (2 strains)

Dipping acetic acid

0.2

Dipping citric acid

0.8

Dipping lactic acid

1.1

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2.2 Plant extracts

The negative side effects of chemical sanitizers together with the growing demand for organic food force the food industry to look for alternative antimicrobials. Besides the organic acids, plant extracts possess a potential antimicrobial activity. The inhibitory effects of different extracts vary depending on the target microorganism and on the type of food. For instance, the hydrophilic cell wall of gram negative bacteria exhibits a greater resistance. The antimicrobial activity of 12 different plant extracts on lettuce against Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes was recently investigated [34]. As conclusion, more than 2 log reductions of S. Typhimurium and E. coli O157:H7 with 5 % of clove extract treatment for 10 min could be achieved and more than 3 log reductions when 10 % of extract is applied for 3 min. It is also pointed out that the sensory quality of lettuce is not affected significantly [34]. Furthermore, studies reported about the application of 0.1 and 1 % of basil methyl chavicol (essential oil of basil) to the fresh cut lettuce reducing the initial count of 105cfu/ml Aeromonashydrophilia below the detection limit [65]. Moreover, Pseudomonasfluorescens could be reduced by 3 log units and also could the total viable count by 2 log cfu/ml for the same application of basil methyl chavicol. The effect of thyme essential oil on the reduction of inoculated E. coli O157:H7 to shredded lettuce was investigated [59]. Thyme essential oil is able to reduce E. coli O157:H7 counts in shredded lettuce by 1.91 and 2.33 log when applied at concentrations of 1 mL/L and 10 mL/L, respectively. The application of natural antimicrobials showed to promote the efficiency of chemical sanitizers. Some pathogens and spoilage bacteria may not be inhibited by legally approved doses for chemical sanitizers. However, the combined use of antimicrobials can increase the efficacy of microbial reduction by synergistic effects [56].

2.3 Protective cultures

Other than synergistic effects of combined antimicrobials, antagonistic effects of microorganisms are studied to suppress the growth of pathogens. It is reported that the growth of Aeromonashydrophilia, L. monocytogenes, SalmonellaTyphimurium, Staphylococcus aureus is inhibited on vegetable salads by certain lactic acid bacteria strains [64]. Bacteriocins produced by Leuconostoc spp. have been studied to control the growthof L. monocytogenes in cut iceberg lettuce without sensory alterations. However, the bioprotective potential of such protective cultures depends on the relative concentration of both, the antagonistic cells and the present pathogens [61]. Moreover, it is reported that washing fresh cut lettuce with a solution containing nisin, coagulin and a nisin–coagulin mixture reduced the viability ofL. monocytogenes by 1.2-1.6 log units. Nevertheless, it has been reported that a bacteriocin treatment is only minimally effective to control the growth of the pathogen during the storage period at 4 °C [6].

3. Physical decontamination methods

3.1 Irradiation

Food irradiation is exposing the food to ionizing radiation that generates free radicals from water by radiolysis. Water molecules lose an electron and produce hydroxyl radicals (OH ֿ◌) and hydrogen peroxide (H2O2) which interfere with the bonds of nucleic acids and cause DNA damage [42].Gamma (γ) radiation applies the photon energy less than 106

kJ/Einstein with a wavelength of less than 0.1 nm whereas x-ray radiation has the photon energy between 106 and 2400 kJ/Einstein with a wavelength of between 0.1 and 50 nm [40]. A low dose irradiation (0.25-1.0 kGy) is proper for vegetable decontamination strategies in order to delay the plant physiological processes [27]. Indeed, EFSA recently reported that doses of 1.0 kGy are tolerated as the maximum level for the use on fruits and vegetables, pointing out that the doses above 0.5 kGy induce the sensorial deterioration of lettuce [13]. In contrast to the decision of EFSA, FDA approved in 2008 the utilization of ionizing irradiation for the iceberg lettuce with the limitation of maximum doses of 4.0 kGy [16]. The ionizing radiation has been suggested as an effective tool to reduce pathogenic microorganisms and parasite populations present on raw fruits and vegetable surfaces [9]. There are limited studies investigating the lettuce decontamination by irradiation processes. Nevertheless, published data reveal promising reductions on pathogens especially for E. coli O157:H7 (see table 6). The maximum allowed limit (1 kGy) may not be sufficient to ensure the low microbial population because it does not penetrate the interior of the irradiation batch [25]. Indeed, it is reported that inner part of the whole lettuce received about 0.2 kGy when 1kGy is applied to the surface [31]. Furthermore, chlorine treatment (200ppm) reduced E. coli O157:H7 populations composed of six strains by 1-2 logs, whereas combined treatment with 0.55 kGy of γ-radiation achieved 5.4 log cfu/g reductions when it is applied to shredded lettuce [17]. Although vegetative cells of pathogenic bacteria and most of the foodborne parasites such as protozoa, cestodes and trematodes are quite sensitive to irradiation, the D10 value to inactivate viruses is more than 10 fold higher [9]. There are limited scientific data available on the safety of irradiated produce. This also results in different consumer perceptions to irradiated products [22]. Additionally, irradiation treatment requires high initial costs and an intensive ongoing management, which results in higher prices of the marketed product. The sensory quality of irradiated food demonstrates differences regarding the

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type of treated food [22]. In the case of lettuce, most of the publications reported an acceptable sensory quality during the whole storage period [25, 42, 46, 70] with an increased antioxidant and phenol level after irradiation [15].

Table 6: Relative efficacy of irradiation strategies for the decontamination of shredded lettuce leaves

Type of microorganism Strategy of irradiation

Application dose (kGy)


Reference

E. coli O157:H7 (C7927, EDL933, 204P)

X-Ray radiation

0.1 1.3 [42] 1 4.4

L. monocytogenes (Scott A, F5069, LCDC 81-861)

0.1 1.6 1 4.1

Salmonella (S. Enteritidis, S. JavianaaS. Montevideo)

0.1 1.0 1 4.8

Shigellaflexneri (ATCC 9199, ATCC 12022)

0.1 1.4 1 4.4

Total aerobic count Gamma radiation

1 2.4 (8th day of storage)

[70]

E. coli O157:H7 (C9490, ATCC 35150, ATCC 43894)

Gamma radiation

0.5 ~ 2.3 [46]

1 ~ 4 Total aerobic count Gamma

radiation 0.5 ~ 3.3 [24]

Yeast count ~1.9 E. coli O157:H7 (E0018, 43894, 932, 994, F4546, H1730)

Gamma radiation

0.53 and 200 ppm chlorine

5.4 [17]

3.2 Ultraviolet (UV) treatment

Another physical preservation method is treating food with UV light irradiation. The UV spectrum is divided into three categories depending on the wavelength. UV-A has the longest wavelength from 315 to 400 nm, UV-B extends from 280 to 315 nm and UV-C (100 to 280 nm) which is also known to be germicidal. Ultraviolet radiation causes membrane depolarization and irregular ionic flow at the cell level, leading to the formation of so-called pyrimidine dimers in the DNA strand. Due to the DNA mutations, transcription and replication of the cell is blocked resulting in cell death [4, 6]. DNA absorbance is maximal across the UV-C, which has the shortest wavelength but highest energy (Miller et al., 1999), thus it is used in food processing for the purpose of non-thermal pasteurization and surface decontamination of food [40]. FDA approved in 2000 the UV treatment as an alternative to thermal pasteurization of fresh juice products. In the mentioned regulation it is pointed out that photochemical changes induced by UV treatment do not have any toxicological significance. On the other hand, UV light is considered by the European Union (EU) as irradiation technology. The irradiation process for food has not been harmonized so far and it is still in discussion which type of food is allowed to be treated by radiation [38]. The inhibition of microorganisms by UV light treatments mainly depends on the dose and the type of microorganism that may have varying resistances due to differing cell wall structures and cell compositions [41]. Although the UV radiation is generally more effective at higher doses, it is reported that it causes respiratory stress on vegetables and as the dose increases, so does the respiration rateof lettuce[4,6]. It is reported that a dose of 7.11 kJ/m2 (the highest dose applied in the experiment)applied on different parts of “red oak leaf” lettuce, induced a softening and increased browning of the product [6]. However, the application strategy should be assessed for certain food matrices as the resulting quality parameters vary by the food and dose level [4]. Regarding the yeast and molds level, the shelf life of minimally processed red oak lettuce is one day longer at doses of 1.18 kJ/m2 and 3 days longer when applied at doses of 7.11kJ/m2 [6]. Also the reduction of lactic acid bacteria counts at higher doses could be demonstrated (2.37 kJ/m2,7.11 kJ/m2). However, it is also stated that UV-C treatment seems to stimulate the growth of lactic acid bacteria on lollorosso lettuce at highest doses (8.14 kJ/m2) [4]. The main disadvantage of the UV-C application is the limited scientific information about the microbial efficacy and sensorial changes of the applied product. Relative low costs, lack of residual component and quick exposure times are denoted as advantages [67].

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Table 7: Relative efficacy of UV treatments (254 nm) for the decontamination of lettuce

Type of microorganism Application dose (kJ/ m2)

Fresh produce type


Reference

Psychrotrophic bacteria

8.14 Whole lettuce (lollorosso)

~ 1.0* [4]

Coliforms

~ 0.6*

Yeast count ~ 0.8* Enterobacteriaceae 1.18 Shredded

lettuce (Red oak)

~ 0.5* [6] 7.11

Total aerobic count 7.11 ~ 0.8* 2.37 ~ 1.1*

* Microbiological reduction compared to untreated control after 9-10 days of storage at 5 °C under passive modified

atmosphere

3.3. Electrolyzed water

Electrolyzed oxidizing water (EOW) has been suggested as an alternative decontamination technique due to its strong bactericidal effect [20]. By electrolyzing the sodium chloride (NaCl) solution, EOW produces an electrolyzed acidic solution containing hypochlorous acid (pH 2-3) at the anode side and electrolyzed alkaline solution containing sodium hydroxide (pH 11-13) at the cathode side [30, 48]. The acidic solution contains 20-60 ppm of free chlorine possessing a strong bactericidal effect against many pathogens including E. coli O157:H7, L. monocytogenes, Salmonella and also spoilage bacteria such as Pseudomonas spp. [33, 48, 52]. Although some color changes on lettuce, as the indication of browning have been observed, it is reported that the sensory quality is not significantly affected [62]. Acidic electrolyzed water (AEW) has the advantage of being more effective than chlorine due to the combination of the low pH value and the high oxidation reduction potential [33, 53]. Besides the AEW, the effectiveness of neutral electrolyzed water (NEW) has been studied recently [2, 23]. NEW is generated similarly to AEW, but has a neutral pH (8.0±0.5) which is accomplished by redirecting amounts of the hypochlorous acid solution from anode to cathode chamber and therefore the application has shown to be less corrosive to the equipment and less irritative to the skin [2]. NEW with approximately 50 ppm of free chlorine has an effect against Salmonella, L. innocua, Erwiniacarotovora and E.coli O157:H7 similar to chlorinated water containing 120 ppm of free chlorine [2]. It is reported that after a 3 min treatment of AEW the microbial level of E. coli O157:H7 is reduced below the detection limit [52]. Additionally, deionized water (DW) treatment followed by AEW reduced SalmonellaTyphimurium below the detection limit after 5 minutes. The same authors compared the efficacies of AEW, Alkaline electrolyzed water (AK-EW), sequential treatment of AK-EW+AEW and DW+AEW and concluded the reduction capacity on 3 pathogens (L. monocytogenes, SalmonellaTyphimurium, E. coli O157:H7) as follows: AEW>DW+AEW≈AK-EW+AEW>AK-EW>DW. The reason of the inconsistency between some publications (see table 8) has been suggested as the differences in the method of inoculation (spray-, spot-, drop-, dip-inoculation), and in the attachment of the microbial cells on the lettuce [33, 37]. Furthermore, it is reported that the initial inoculation amount has an effect on the reduction of microorganisms, especially on E. coli. The reduction by 1.6 log cfu/mL after the treatment of NEW containing 52 ppm free chlorine has been investigated, after the lettuce has been dipped into a mixture of three strains (107 cfu/mL Salmonella, E. coli, L. innocua). However, only 1 log reduction could be obtained after it is dipped into the mixture of 105cfu/mL [2]. AEW has the advantage of being more effective than chlorine solutions due to the combination of low pH value and the high oxidation reduction potential. Additionally, it is generated from the NaCl solution on-site and therefore it does not pose danger for handling or storage practices, as well as adverse impacts on the environment [33, 53].

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Table 8: Relative efficacy of electrolyzed water treatments for the decontamination of lettuce products

Type of Microorganism

Application strategy

Amount of free chlorine (ppm)


Fresh produce type


Reference

E. coli O157:H7 (ATCC 35150, ATCC 43889, ATCC 43890)

AEW1)

pH 2.6

37.5±2.5

1

Shredded lettuce

3.5 [53]

S. Typhimurium (ATCC 19585, ATCC 43174, ATCC 363755)

3.4

L. monocytogenes (ATCC 19113, ATCC 19114, ATCC 7644)

3

L. monocytogenes (ATCC 19117, 109, 201, 315,116)

AEW1) pH 2.5

45 1 Whole lettuce (iceberg)

2.6 [52]

E. coli O157:H7 (932, 994, E0018, H1730, F4546)

2.4

E.coliO157:H7 (SEA 13B88)

AEW1) pH 2.6

50 2 Shredded lettuce (iceberg)

0.7 [33] 20 1.0

Total aerobic count AEW1) pH 2.6

30 10 Whole lettuce

~ 2.3 [36] Mold and Yeast count ~ 1.5 Coliforms ~ 2.0 Total aerobic count

NEW2)

pH 8.6

52±6

3

Shredded lettuce (iceberg) Inoculated by dipping 108cfu/ml

0.8 [2]

E. coli O157:H7 (NCTC 12900)

48±4

~1.2

Salmonella enterica serotypechloreaesuis

~1.4

L. innocua (CECT-910)

~1.5

Erwiniacarotovora ssp. carotovora CECT-225

~ 1.2

1)Acidic Electrolyzed Water 2)Neutral Electrolyzed Water

Discussion and Conclusion

Fresh vegetables are one of the essential constituents of the human diet. There is a relatively large number of evidences associated with their nutritional and health benefits regarding vitamin, mineral and dietary fiber content [1, 2, 3, 17, 34, 35, 42, 52,56, 67]. Among the minimally processed vegetables (MPV), lettuce is one of the most popular vegetables that is eaten without heating or cooking [1, 34]. Since MPV are not exposed to intense preservation treatments, it is difficult to maintain the safety and quality aspects during production [35]. Hence, safety concerns emerge the necessity to deal with the decontamination methods ensuring the safety of the fresh vegetables. Among the decontamination strategies for vegetable processing, chlorine solutions containing between 50-200 ppm free chlorine are the most commonly used commercial sanitizers [51, 55, 59]. However, the potential health impacts and environmental effects regarding the by-products generated from the reaction with organic matters should be considered [21, 55]. Additionally, its effect on microorganisms is regularly limited by 1-2 log reductions (see table 1). Chlorine dioxide, the alternative to chlorine solution possesses comparable microbicidal decontamination properties (see table2). Hydrogen peroxide showed unsatisfactory results due to sensorial product alterations [51]. On the other hand, aerosolized peroxyacetic acid has been associated with more than 3 log reductions on SalmonellaTyphimurium, with the application time of 30 min (see table 3) [47]. Physical preservation of food by UV-treatment is an approved technology in the U.S. (FDA, 2000). However in the European Union this technology is considered as irradiation treatment and therefore harmonization for food approvals are still in progress. Gamma irradiation demonstrated a valuable microbial reduction efficiency (4 log cfu/g) on pathogens, such as E.coli O157:H7, and Shigellaflexneri(see table 6). However, product softening should be

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encountered when doses of 1 kGy are applied. Studies on the effectiveness and sensorial impacts on fresh-cut lettuce by UV-C treatment are still underrepresented. Ozone with its strong oxidation-reduction potential has been used for water treatment for many years [21]. Bubbling ozone treatment showed increased effects on microbial reduction due to mechanical forces weakening the attachment of cells on vegetable surfaces (see table 4) [50]. Organic acid treatments demonstrate varied results ranging from 0 to 3 log reductions (see table 5). Lactic acid and citric acid can be considered more effective than acetic acid for the fresh-cut lettuce decontamination. Fumaric acid treatment (50mM) has been reported to be more effective than 200 ppm NaClO treatment, with a 1.4 log reduction on the microbiota (see table 6). However, browning effects on the product after treatment could not be eliminated [34]. Combinations of chlorinated water, peroxyacetic acid, citric acid and water to reduce L. monocytogenesin lettuce demonstrated different recoveries for certain strains. L. monocytogenes isolates obtained from lettuce were highly recoverable [26]. In general, electrolyzed oxidizing water (EOW) also demonstrates proper reduction results with minimal negative effects on the product and human health issues. Nonetheless, some published trials indicate inconsistency in their results (see table 8), probably due to the strain adaption to acidic environments. In this review, the decontamination methods with different agents (chemical and natural origin) associated with fresh-cut lettuce are discussed and their specific strengths and weaknesses are outlined. Through the investigation of available scientific data, it can be concluded that the microbial reduction on (fresh-cut) lettuce depends on various influence criteria as follows: the applied technology, temperature and duration of treatment, pH value and concentration, type of microorganism and strain specific characteristics. Moreover, it can be asserted that the state of attachment of a certain microorganism to the surface of the lettuce has a critical effect on the actual microbial reduction. The state of attachment is interconnected with the incubation time of the inoculated target microorganism [34]. Legal issues regarding the approval status of agents should be carefully inspected before implementing them in daily manufacturing practice. Risk evaluations are still ongoing, as the recently published opinion on the irradiation of food by the European Food Safety Authority [13]. In summary, being able to recommend a superior method on the basis of scientific literature, there is still a workload outstanding to optimize method parameters to obtain reproducible treatments for the generation of microbiologically safe, residue-free and acceptable fresh-cut produce.

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