Contemporary strategies in combating microbial contamination in food chain

International Journal of Food Microbiology 141 (2010) S29–S42

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Review

Contemporary strategies in combating microbial contamination in food chain

Andreja Rajkovic ⁎, Nada Smigic, Frank DevlieghereLaboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Food2Know, Ghent University, Ghent, Belgium

⁎ Corresponding author. Laboratory of Food MicrobDepartment of Food Safety and Food Quality, Faculty ofUniversity, COUPURE LINKS 653, B-9000 Ghent, Belgium+32 9 225 55 10.

E-mail address: [email protected] (A. Rajk

0168-1605/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2009.12.019

a b s t r a c t
a r t i c l e i n f o
Keywords:
DecontaminationPreservationHurdlesVirulenceResistanceFood
The objective of this review has been to disclose collected information on benefits and risks of selected “less-than – sterilizing” processes applied to control microbial hazards in food that was meticulously collected andcritically reviewed during five years of EU Sixth framework project “Pathogen Combat”. The target organismsof the project, and thus of this review, too, were Listeria monocytogenes, Escherichia coli O157:H7 andCampylobacter jejuni. Due to their specific response and high relevancy to the food safety, foodborne virusesand spores, were also discussed within the scope of this review. Selected treatments comprised HighPressure Processing, Intense Light Pulses, treatments with organic acids, treatments with chlorine dioxideand for their relevancy also mild heat treatments and Pulsed Electric Field processing were included. Themain aspects included in this review were principles of the processes used and their application, sub-lethalinjury and its consequences on microbial food safety, and legal platform and its impact on wide use of thetreatments. Finally a reflection has been made to combined application of different hurdles andaccompanying risks.

iology and Food Preservation,Bioscience Engineering, Ghent. Tel.: +32 9 264 60 85; fax:

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S292. Mild heat treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S303. High pressure processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S314. Pulsed electric fields (PEF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S335. Intense light pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S346. Weak organic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S367. Aqueous chlorine dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S378. Food preservation by combined processes (hurdle technology) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S389. The risks to be considered . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S38References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S39

1. Introduction

Number of alternative methods and technologies rose up to replacehistorically proven heat treatments in attempt to satisfy modern trendsin food consumption. These new trends were induced by the change inthe consumers' perception of food quality and nutrition. The modern

consumer seeks fresh looking, convenient and nutritionally healthyfood. This requires from industry to adopt new strategies in safe foodproduction, using sustainablemethodswith small carbon footprint. Themain change in terms of microbial food safety is that sterilization andpasteurization as we knew them are in great extent replaced by mildheat treatments, high pressure processing, pulsed electricfields, intenselight pulses, applicationof organic acids, chlorinedioxide, etc. The abilityof these technologies, alone or in combination to inactivate micro-organisms, is beneficial for the applications in heat sensitive foods andingredients and for minimization of adverse effects on the sensorycharacteristics of food products. Many of these novel technologies havebeen alreadysubjectof extensive research, but before actual commercialapplication takes place the number of technical, economical, and

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Table 1Percent of (n=198) respondents that were “very” or “extremely” concernedwith foodsprocessed by novel food processing techniques (adopted from Wright, Cardello, & Bell,2007).

Food processing method % Very or extremely concerned % Uncertain

Genetic modification 54 17Irradiation 49 17Radio frequency sterilization 40 21High pressure treatment 20 18Microwave processing 18 12Thermal processing, general terms 18 14Heat pasteurization 13 6

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regulatory issues are to be solved. Among the factors thatwill determinethe success of certain novel technology is the consumers' acceptance ofthe food product. Although many consumers prefer the non-thermalfood processing technologies to manufacture higher quality, value-added foods that feature higher vitamin and nutrient retention and theimproved sensory attributes, the lack of knowledge may pose anobstacle in their buying behaviour (Table 1).

The ideal processing technologies for the food producers would bethe one that meets the following demands (taken from Raso, Pagan, &Condon, 2005):

• Improvement of the shelf life and safety by inactivating enzymes,spoilage and pathogenic microorganisms

• No changes in organoleptic and nutritional attributes• No residues left on food• Convenient to apply• Cheap• No objections from consumers and legislators

In food safety, or even broader in food processing terms, “novel”inactivation technologies (intervention) have to be described in theirequivalents to the heat sterilization and pasteurization. While heattreatments are in the termsofmicrobial inactivation characterizedusingvalues such as D value (decimal reduction time, is the time of the heattreatment in minutes which gives an expectedmicrobial inactivation of90%), Z value (temperature change required for 10-fold change in Dvalue) and F value (the reference time in minutes at the referencetemperature of 121 °C for thedesired sterilization effect), the equivalentparameters need to be described for non-thermal processing.

Over the years, different studies have demonstrated the success andfailures of mild processing technologies in the inactivation of initialmicrobial load in food. For many of these technologies the potential toproduce high quality foods that are microbiologically safe within theextended shelf-life was demonstrated. However, reports of ambiguousfindings leave certain puzzlement in data interpretation. Applications ofmolecular techniques and studies on intracellular level have recentlybrought new insights, providing evidence of variation in microbialresponse to sub-lethal treatments (Shabala, McMeekin, Budde, &Siegumfeldt, 2006; Smigic et al., 2009b). The threat of modifiedproperties of surviving pathogens necessitates evaluation of the micro-organisms under such circumstances, with special attention given to theeffect of variability at single cell level for pathogens with low infectivedose (Francois, Devlieghere, Uyttendaele, & Debevere, 2006).

It has been our intention to collect and filter fragmented informationregarding microbial behaviour in the response to pulsed electric field,high hydrostatic pressure, intense light pulses, chlorine dioxide andorganic acids as an example of alternative decontamination treatmentsand allow deduction of advantages and dangers of their application.

2. Mild heat treatments

Heat is the most commonly used preservation method in foodprocessing, and heat-treated foods generally have a good safety

record. When properly applied, heat can eliminate biological agentsthat spoil or compromise food safety. The applied treatment factors(time/temperature regime) can vary to accomplish almost any degreeof microbial inactivation, ranging from limited reductions of microbialload to complete sterilization (Juneja and Novak, 2003; Yousef andCourtney, 2003). Heat causes damage to macromolecular cellcomponents; thus the main function of heat-induced stress proteinsis to repair or destroy these damaged components so that they do notdisrupt cellular metabolism. Many heat-induced stress proteins areprotein chaperones that assist in folding and assembly of heat-damaged proteins (Yousef and Courtney, 2003). In addition to thesechanges, some bacteria also alter their cell membrane in response toheat by increasing the ratio of trans to cis fatty acids in the membrane.This structural change is thought to decrease fluidity caused byincreasing temperatures (Cronan, 2002). Nguyen, Corry, and Miles(2006) showed that cell death in Campylobacter jejuni and Campylo-bacter coli coincided with unfolding of the most thermally labileregions of the ribosome. Moreover, alpha and beta subunits of RNApolymerase, might also unfold at the same time and contribute to celldeath.

Differences in the reported data regarding heat-induced sub-lethalinjury might be due to different time/temperature regimes used (Buschand Donnelly, 1992; Jasson, Uyttendaele, Rajkovic, & Debevere, 2007;Uyttendaele et al., 2008). The results obtained by Uyttendaele et al.(2008) and Jasson et al. (2007) indicated no sub-lethal injury in Listeriamonocytogenes cultures treated with 60 °C for 20 min. However, highpercentages of sub-lethal injury in L. monocytogenes and Listeria inoccua(98.1 to 99.9%) were determined in the study of Busch and Donnelly(1992) when exposed to 56 °C for 50 min. Mild heat treatmentcomprised of 56 °C for 5 min resulted in 75.7 and 85.8% of sub-lethalinjury in two different Escherichia coli O157:H7 strains (Jasson et al.,2007). Similar, high level of injury was also determined in mild heattreated cultures of Salmonella Typhimurium (Wuytack et al., 2003). Theresearchers investigated the potential for heat treated cells to survive/grow under suboptimal storage conditions. Van Houteghem et al.(2008) determined no difference between heat-treated and untreatedcultures of L. monocytogenes when stored under CO2 rich atmospheres.This was expected, as the respective heat treatment did not induce anysub-lethal injury in the cell cultures. However, several other reportsspecified that sub-lethally heat injured L. monocytogenes, E. coli O157:H7 and Aeromonas hydrophila showed reduced ability to grow/surviveadverse storage conditions (Golden, Eyles, & Beuchat, 1989; Semanchek,Golden, & Williams, 1999; Williams and Golden, 2001), revealing thatmild heat treatment might be used in combination with otherpreservation factors for controlling foodborne pathogens in foods.

It is of note that pre-exposure to other stresses, as part of themicroorganism's history, can be of an impact on the heat resistance.Skandamis, Yoon, Stopforth, Kendall, and Sofos (2008) found thatcombinations and sequences of sub-lethal hurdles may affect L.monocytogenesheat tolerance. The highest heat resistancewasobservedafter combined exposure to acid andheat shock followed byexposure toosmotic shock, and by the combination of osmotic with heat shock. Thesequence of exposure to sub-lethal stresses did not affect thethermotolerance of L. monocytogenes, whereas simultaneous exposureto multiple hurdles resulted in higher survivors of L. monocytogenes.

Baert, Uyttendaele, Van Coillie, and Debevere (2008) reported 1.86,2.77, and 3.89 log reduction of, respectively murine norovirus 1(MNV-1), B. fragilis infecting phage and E. coli after a mild thermalpasteurization of raspberry puree. Heating at 75 °C for 15 s established2.81 log reduction of MNV-1 while 3.44 and 3.61 log reduction of B40-8 and E. coli was observed. Comparison of feline calicivirus (FCV) andcanine calicivirus (CaCV) indicated similar inactivation rates attemperatures ranging from 37 °C to 100 °C (Duizer et al., 2004). Similarthermal inactivation rates at 63 °C and 72 °Cwere observed for FCV andmurinenorovirus1 (Cannonet al., 2006).At 71 °C, exposure of 0.16, 0.18and 0.52 minwere needed in respectively skimmedmilk, homogenized

Table 2Overview of requirements for cook-chill, cook-freeze and sous-vide foods (James andJames, 2005).

Stage in process Requirements

Cook-chillInitial cooking 70 °C for not less than 2 minMinimum time for chilling tobegin after initial cooking cycle

30 min

Chilling time 1.5 h to 3 °C2.5 h or sooner for larger meatsMeat joints are recommended not to exceed3 kg or 100 mm thick

Storage temperature 0 °C to 3 °C with 0.2 °C toleranceShelf-life at storage temperature 5 daysCritical temperature during storage 5–10 °C, and consume within 24 h

Above 10 °C destroyRe-heating temperature Minimum 70 °C for not less than 2 min

Cook-freezeInitial cooking 70 °C for not less than 2 minMinimum time for chilling tobegin after initial cooking cycle

30 min

Freezing time 1.5 h to 5 °C at centreStorage temperature 18 °CShelf-life at storage temperature In general up to 8 weeks without significant

changesCritical temperature duringstorage

Partly/completely thawed food not to berefrozen.Food thawed at unknown temperatures not tobe consumed

Re-heating temperature Minimum 70 °C for not less than 2 min

Sous-videInitial cooking minimum 70 °C, time not specifiedPortioning 10 °C within 30 minChilling time 1.5 h to 3 °CStorage temperature 18 °CShelf-life at storage temperature In general up to 8 weeks without significant

changesCritical temperature duringstorage

5 °C–10 °C and consume within 12 hAbove 10 °C destroy

Distribution For short periods insulated containers suffice.For longer periods, refrigerated transport isrequired

Re-heating temperatureminimum

70 °C

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milk and creamto reduceHepatitis A virus (HAV) by1 log,whereas4 logreduction required 6.55 (skim), 8.31 (homogenized) and 12.67 (cream)min (Bidawid, Farber, Sattar, &Hayward, 2000).A longerheat treatmentwasneeded in creamto achieve similar inactivation ofHAV compared tomilk. The same authors noted a nonlinear decline of HAV in milkbetween 65 °C and 75 °C, where the rapid inactivation in the initialphase could be explained by freely suspended virus particles incomparison to viral aggregates.

Bacterial spores are characterized by higher heat resistance incomparison to vegetative cells (e.g. vegetative cells of B. cereuswill bedestroyed by heat treatment of 20 min at 70 °C, while spores willmainly remain unharmed, Faille, Lebret, Gavini, &Maingonnati, 1997).Among the most important spores in terms of public health are thoseof non-proteolytic Clostridium botulinum (psychrotrophic, producetoxins of types B, E, or F, and are capable of growth and toxinproduction at 3.3 °C), proteolytic C. botulinum (mesophilic producetoxins of type A, B and F with spores more heat resistant than those ofnon-proteolytic strains), Bacillus cereus (psychrotrophic and meso-philic), and Clostridium perfringens (mesophilic). It is important tonote the psychrotrophic nature for some of these pathogens, whichmakes them an ultimate danger in foods that undergo only mild heattreatment and that rely on a cold-chain for their preservation.

A critical application of mild heat processing is in the production ofmodern refrigerated processed foods of extended durability (REPFED)where the crucial microbiological safety and quality considerations isthat all the components that form the complete ready meal have beenprocessed in a manner that destroys non-sporeforming pathogenicmicroorganisms. REPFEDs are processed at a lower temperature withmaximum within the range of 65–95 °C (Peck, 1997). Examples ofREPFEDs are sous-vide, cook-chill and cook-freeze foods (James andJames, 2005). The heat process is intended tomaximize the sensory andorganoleptic qualities of products whose characteristics would beadversely affected by heating at a higher temperature. For example inUK food safety agency guidelines stipulate that cooking food until thecore temperature of 75 °C or above will ensure that pathogens aredestroyed. However, lower cooking temperatures are acceptableproviding that the core temperature ismaintained for a specified periodof time (http://www.food.gov.uk/multimedia/pdfs/csctcooking.pdf):

• 60 °C for a minimum of 45 min• 65 °C for a minimum of 10 min• 70 °C for a minimum of 2 min.

After heat treatment products are cooled rapidly, and stored atrefrigeration temperatures. REPFEDs are generally prepared in one ofthree ways: vacuum packaged ingredients are cooked (e.g. sous-videfoods); ingredients are cooked individually and then packaged; oringredients are cooked, packaged and then heated again (Peck, 1997).To avoid contamination of the cooked product, some systems hot-fillthe packs and seal them before cooling and there are a few systemswhere the raw ingredients are assembled, sealed in the pack and thepack cooked and cooled. Once the meal has left the factory the extentof bacterial growth is primarily a function of the product time/temperature history and presence of preservatives. Clearly, the lowerthe initial contamination of the ingredients the better the outcome ofthe overall preservation. Therefore, more than one control measuremay be required to control an identified hazard, e.g. temperature, pH,moisture content, preservative or salt level and gas concentration inmodified atmosphere package (Rajkovic et al., 2009b).

Table 2 shows current requirements for safe application of cook-chill food production. Scientific data indicates that a temperature of70 °C for 2 min or equivalent, at the core of a food, is sufficient toensure a 6 log reduction in numbers of L. monocytogenes beingconsidered the most heat resistant vegetative pathogen if present(Gaze, Brown, Gaskell, & Banks, 1989).

In the chilling stage a fast passage through the area of temperaturebetween 55 °C and 15 °C is a basic demand for microbial safety of the

cook-chill products (Kleiner and Motsch, 1999). It is recommendedthat temperature of 3 °C is reached within 1.5 h. At 3 °C the lag phaseof the majority of non-spore forming psychrotrophic pathogenicbacteria, such as L. monocytogenes, is about a week with 20 hgeneration time afterwards (Mossel and Struijk, 1991). Consequently,a product contaminated with 10 CFU/g of L. monocytogenes, forexample, will reach European legal limit of 100 CFU/g (EU, 2005)within about 10 days of storage. Any abuse of temperature control inthe preparation, storage, distribution and reheating may result inpublic health hazards.

3. High pressure processing

High pressure processing (HPP) has the potential to deliver athermal equivalent of pasteurization or sterilization with microbialreductions in process time without appreciable changes in colour,flavour, and texture. The effects of high pressure are instantaneousthroughout a food product. The inactivation mechanism of HPPproceeds through low energy and does not promote formation ofunwanted new chemical compounds, or free radicals that can resultwhen foods are, for example, irradiated. The effects of HPP onmicrobial food safety and quality have been expressively summarizedelsewhere (Cheftel, 1995; Considine, Kelly, Fitzgerald, Hill, & Sleator,2008).

http://www.food.gov.uk/multimedia/pdfs/csctcooking.pdf


The commercial production of pressurized foods has become areality in Japan, France, Spain, the USA and many other countries. Thisis a result of an extensive scientific research, technological andtechnical advances in HPP equipment production and decrease in theprocessing costs (Cheftel and Culioli, 1997). Nowadays, a commercialapplication of high hydrostatic pressure has found its place in theproduction of juices, sauces, smoothies, ready-to-eat meat products,guacamole, oysters etc (Hjelmqwist, 2005; Patterson, Linton, & Doona,2007). An important advantage of HPP in comparison to many otherintervention technologies is that food items can be subjected to highpressure with or without packaging, which in former case eliminatesthe possibility of post-treatment contamination.

A typical high pressure system for food processing consists of apressure vessel in which food packages are loaded and into which thepressure medium, usually water, is pumped, and a pressure-generatingdevice. In the case of liquids, such as fruit juices, the vessel is filled withthe juice,which acts as the pressure transmissionfluid. Once the desiredpressure is reached, the pressure can be maintained without furtherneed for energy input. A fundamental principle underlying HPP is theisostatic process allowing all regions of the food to be rapidly exposed toa uniformpressure. Thework of compression duringHPP treatment alsoincreases the temperature of foods through a process known asadiabatic heating, and the extent of the temperature increase varieswith the composition of the food (usually 3–9 °C/100 MPa). HPP istraditionally a batchprocess, and this is the only system that can beusedfor solid foods. However, liquid foods can be treated in a series of vesselswhich can work in a staggered sequence for an overall system that issemi-continuous (Considine et al., 2008).

The application of pressures in the range of 300–600 MPa, applied atambient temperature within a few minutes, should result in aninactivation of vegetative cells of key foodborne pathogens (Table 3).HPP treatment is generally considered toaffectbacterial cellmembranesand impair their permeability and ion exchange, but also to inactivatesomeof the enzymes vital for survival and reproduction of bacterial cells(Cheftel, 1995; Hoover, Metrick, Papineau, Farkas, & Knorr, 1989;Yaldagard,Mortazavi, & Tabatabaie, 2008).Microorganisms varywidelyin their resistance to HPP treatment with some strains being moreresistant than other (Alpas, Kalchayanand, Bozoglu, & Ray, 1998; Chungand Yousef, 2008). This could be the reason for great variation inreported results obtained by researchers using different strains of thesame species. Approximately 3 log cycles difference in viability loss was

Table 3The efficiency of high pressure processing (HPP) against E. coli O157:H7, L. monocytogenes and

Pathogen Food product Treatment conditions

E. coli O157:H7 Apricot juice (pH 3.8) 250 MPa, 5 min, 30 °COrange juice (pH 3.76) 250 MPa, 5 min, 30 °CSour cherry juice (pH 3.3) 250 MPa, 5 min, 30 °CApple juice (pH 3.5) 500 MPa, 5 min, 20 °CTomato juice (pH 4.1) 500 MPa, 5 min, 20 °COrange juice (pH 3.8) 500 MPa, 5 min, 20 °CRaw minced meat 700 MPa, 1 min, 15 °CHungarian salami 600 MPa, 6 min, 25 °C

L. monocytogenes Human milk 400 MPa, 1.5 min, 31 °CTurkish white cheese 600 MPa, 5 min, 25 °CRaw milk 500 MPa, 10 min, 20 °CFish slurry 400 MPa, 5 min, 20 °C

C. jejuni UHT whole milk 325 MPa, 10 min, 25 °CUHT skim milk 325 MPa, 10 min, 25 °CSoya milk 325 MPa, 10 min, 25 °CChicken puree 325 MPa, 10 min, 25 °CPhosphate buffer 325 MPa, 10 min, 25 °CMilk 300 MPa, 10 min, 20 °CBroth 300 MPa, 10 min, 20 °CChicken meat slurry 200 MPa, 10 min, 20 °C

a Results obtained in different strains.

reported for different strains of L. monocytogenes and E. coli O157:H7when exposed to 345 MPa for 5 min at 25 °C, and even 7 log cyclesdifference for Staphylococcus aureus strains exposed to the same HPPtreatment (Alpas et al., 1999). Beside intrinsic barotolerance, Haubenet al. (1997) reported induced barotolerance determinedwhen alteringcycles of HPP treatments and growth were applied in cultures of E. coli.Selected pressure resistant mutants were also more heat resistantcompared to parental strains, revealing that barotolerance should beproperly understood to predict safety of HPP treated foods (Haubenet al., 1997). As it will be seen later the same phenomena has beenobserved for several other technologies.

Not only bacterial inherent characteristics and treatment para-meters are determining the effectiveness of HPP, but also theenvironment in which bacteria are found. Patterson, Quinn, Simpson,and Gilmour (1995) reported that treating E. coli O157:H7 with700 MPa for 30 min at 20 °C resulted in 6 log reduction in phosphate-buffered saline, 4 log reduction in poultry meat, and <2 log reductionin UHT milk. The reason might be probably found in the protectiverole of certain food constituents. The intrinsic characteristics of food,such as pH and water activity (aw) might also affect the inactivationefficiency of HPP. Most microorganisms are more susceptible to highpressure in lower pH environments, and even more pressuredamaged cells are less likely to survive subsequent storage in acidicenvironments (Linton, McClements, & Patterson, 1999; Pagan, Jordan,Benito, & Mackey, 2001; Patterson et al., 2007).

The subpopulation of sub-lethally injured cells might occur incultures treated with HHP, although the percentage of injured cellshighly depends on the treatment conditions used. The increase in timeof the exposure, temperature and pressure, resulted in the greaterpercentage of sub-lethally injured cells (Alpas et al., 1998). Bozoglu,Alpas, and Kaletunc (2004) reported that pressure damaged cells of L.monocytogenes, S. aureus, E. coli O157:H7 and S. enteritidis were able torecover during subsequent storage in milk. The possibility of pressuretreated cells to repair during storage has to be taken into considerationfor low acidic foods, as their safetymight beoverestimated. On the otherhand, the injury caused by HPP can be advantageous for high acidicfoods, as it is known that pressure treated cells are more susceptible toacidic environment (Linton et al., 1999). These results highlight theimportance of combining pressure processing with other hurdles, suchas acidic environment or low temperature, which should preventrecovery of pressure injured cells.

C. jejuni.

Log reduction Reference

4.85 BayIndIrlI, Alpas, Bozoglu, and HIzal (2006)5.1 BayIndIrlI et al. (2006)5.28 BayIndIrlI et al. (2006)5 Jordan, Pascual, Bracey, and Mackey (2001)5 Jordan et al. (2001)1–2 Jordan et al. (2001)5 Gola, Mutti, Manganelli, Squarcina, and Rovere (2000)>4 Gill and Ramaswamy (2008)≈6 Viazis, Farkas, Jaykus (2008)4.3–4.4 Evrendilek, Koca, Harper, and Balasubramaniam (2008)>4 Linton, Mackle, Upadhyay, Kelly, and Patterson (2008)≈3 Ramaswamy, Zaman, and Smith (2008)≈2.5 Solomon and Hoover (2004)≈2.5 Solomon and Hoover (2004)≈3 Solomon and Hoover (2004)≈3.5 Solomon and Hoover (2004)8 Solomon and Hoover (2004)0.4–1a Martinez-Rodriguez and Mackey (2005)3–6.7a Martinez-Rodriguez and Mackey (2005)0.2–2.2a Martinez-Rodriguez and Mackey (2005)


The inactivation of spores by HPP is less efficient compared toefficiency in vegetative cells, and requires higher pressures and highertemperatures (Heinz and Knorr, 2005 and references therein). Bacterialspores were found to survive up to 1200 MPa at room temperature(Zhang andMittal, 2008 and references therein). Furthermore, a detailedreview of Zhang et al. (2008) compiled much of the published datashowing that there can be significant variations in the requirements ofhigh pressure and temperature among different bacterial spore speciesand also among strains of the same species. The optimization of the HPPconditions or combination with other treatments and agents may beneeded for a successful inactivation of spores. Most of the time theinactivation of spores is a two-step process: first, germination of thespores, and second, subsequent inactivation of the germinated spores.Germination is the process by which a stimulus is applied to induce thedormant spores to convert to ametabolically active vegetative state. Heatshock is probably the most common stimulus. In principle, if all of thespores present in a foodmaterial could be induced to germinate, the foodmaterial could then be sterilized by a subsequent treatment that wouldbemilder than the treatment needed to inactivate ungerminated spores.

Regarding inactivation of foodborne viruses, differences inHPPeffectwere noticed between different viruses, different treatment parametersand different foods/media (Baert, Debevere, & Uyttendaele, 2009;Kingsley, Hollinian, Calci, Chen, & Flick, 2007). In review of Baert et al.(2009) it was reported that exposure of hepatitis A virus to pressures of375 MPa for 5 min at 21 °C induced reduction of respectively 4.3 and 4.7log in strawberry puree and on sliced green onions, respectively. HPPtreatment of oysters with a pressure of 400 MPa for 1 min (9 °C)induced 3 log reduction of HAV, whereas MNV-1 was reduced by 4 log(5 °C). On the other hand, Aichivirus and coxsackievirus B5 remainedfully infectious if 600 MPa was applied for 5 min at ambient temper-aturewhereas coxsackievirus A9was reduced by7.6 log under the sameconditions. Similarly polioviruswas found to be resistant to 600 MPa for1 h. It can be concluded that the sensitivity towards HPP does not agreebetween genetically related taxonomic groups or even strains. Apossible explanation could be the difference in protein sequence andstructure (Baert et al., 2009).

In EU countries, the legislation of high pressure processed food isincorporated in the Novel Food Regulation (EC) No 258/97. In 2001,the EU authorized the Group Danone to launch high pressurepasteurised fruit-based preparation with the Decision 2001/424/EC.In the United States the traditional health regulations are applied andproducts treated by HP, such as guacamole, ham, fruit juices, salsadips, RTE foods and oysters, have already been introduced to themarket without any specific regulation. Number of HHP-commerciallyproduced foods is available in Japan, too.

4. Pulsed electric fields (PEF)

Technology of pulsed electric fields (PEF) is a non-thermal inactiva-tion technology based on the use of electric fields of high voltage. Next tothe microbial inactivation, PEF maintains food quality attributes such as

Table 4The efficiency of pulsed electric fields (PEF) treatment against E. coli O157:H7 and L. monoc


E. coli O157:H7 Apple cider 80 KV/cm, 30 pulses, 2 µs/pulse, 42 °CApple juice 34 KV/cm, treatment time 166 µs, 4 µs/pulse, 30 °CApple juice 31 KV/cm, treatment time 202 µs, 4 µs/pulse, 30 °CSkim milk 24 KV/cm, treatment time 141 µs, 2.8 µs/pulse, 30 °Liquid egg yolk 30 KV/cm, 105 pulses, 2 µs/pulse, 40 °CEgg white 15 KV/cm, 500 pulses, 0 °CEgg yolk 15 KV/cm, 500 pulses, 0 °CWhole egg 15 KV/cm, 500 pulses, 0 °C

L. monocytogenes Melon juice 35 KV/cm, treatment time 2000 µs, 4 µs/pulse, 39 °Watermelon juice 35 KV/cm, treatment time 2000 µs, 4 µs/pulse, 30 °Skim milk 20 KV/cm, 10 pulses, 3.25 µs/pulse, 35 °CWhole milk 30 KV/cm, 400 pulses, 1.5 µs/pulse, 50 °C

sensory, quality and nutritional value. The application of PEF is restrictedto the foods that can sustain high electric fields, have low electricalconductivity and do not have or produce bubbles. Therefore, successfulapplication of PEF technology to liquidproducts such as fruit juices, liquideggs, fruit smoothies and milk (Jin, Zhang, Hermawan, & Dantzer, 2009;Marselles-Fontanet, Puig, Olmos, Minguez-Sanz, &Martin-Belloso, 2009;Riener, Noci, Cronin, Morgan, & Lyng, 2009; Walkling-Ribeiro, Noci,Cronin, Lyng, Morgan, 2009; Walkling-Ribeiro, Noci, Cronin, Lyng, &Morgan, 2008) at laboratory and pilot plant levels suggests the potentialof this technology as a substitute for traditional thermal pasteurization.

A typical batch PEF system consists of a high-voltage pulse generatorand a treatment chamber. Additional devices for degassing, vacuuming,preheating, and cooling of treatmentmedium, can be added. PEFmay beapplied as oscillating, bipolar, exponentially decaying or square wavepulse. Bipolar waves are more lethal to microbial cells compared tomonopolar, as rapid changes in movement direction can easier damagemicrobial cell membrane. The equipments that are mostly in use aredesigned to deliver filed strength from 20 till 70 KV/cm, with pulsedurationbetween1and5 µs. Repetitionof thepulses is set upbetween1and 30 s, to avoid overheating of the sample when higher voltages areused. The application of PEF to liquid foodsmaybeoperated as a batchorcontinuous system.When the batch operation is applied, food is placedin the PEF chamberwhere the high voltage electric pulses are delivered.When continuous system is used, liquid food runs continuously throughthe PEF chamber where the pulses are applied (Gould, 2005). Forcommercial PEF treatment plant one of the major factors affecting PEFsystem design is flow rate of fluid foodstuff. The flow rate determinesseveral major PEF system characteristics, such as the pipe diameter, theaverage power required for a given fluid etc. Nowadays, for sterilizingand pasteurizing foods, packaging, and other purposes, advancedsystems are developed and commercialized by Maxwell's PurePulseTechnologies Inc. subsidiary, whose CoolPure™ PEF system is used forreduced-temperature pasteurization of different liquid, as well ascheese, with the treatment velocity of 10 and 200 L/h, respectively(Huang and Wang, 2009).

PEF has been reported to inactivate wide range of foodbornepathogens, including E. coli O157:H7, Salmonella, L. monocytogenes, S.aureus (Barbosa-Canovas and Sepulveda, 2005). Table 4 gives anoverview of several studies taken to investigate the effect of PEF on E.coli O157:H7 and L. monocytogenes in different foods. The exactmechanism by which PEF inactivate microorganisms is not completelyunderstood. However, much of the research in the field points towardperturbation of the cell membrane and loss of membrane permeabilityas principal factors responsible for the microbial inactivation (Heinz,Alvarez, Angersbach, & Knorr, 2001). The alteration in the ion transportand conformational changes of the microbial enzymes might also occurwhen PEF is applied to microbial cells (Góngora-Nieto et al., 2002).Other effects resulting from the application of high-intensity pulsedelectric fields, such as DNA damage and generation of toxic compounds,have been also suggested, although some of the later studies rejectedthese hypotheses (Barbosa-Canovas and Sepulveda, 2005). Even though

ytogenes.


5.35 Iu, Mittal, and Griffiths (2001)≈4 Evrendilek et al. (2000)2.56–1.63 Evrendilek and Zhang (2005)

C 1.27–1.88 Evrendilek and Zhang (2005)4.9 Amiali, Ngadi, Smith, and Raghavan (2007)≈1 Amiali, Ngadi, Raghavan, and Smith (2004)≈3 Amiali et al. (2004)≈3.5 Amiali et al. (2004)

C 4.27 Mosqueda-Melgar, Raybaudi-Massilia, andMarton-Belloso (2007)C 3.77 Mosqueda-Melgar et al. (2007)

1 Fleischman et al. (2004)4 Reina et al. (1998)


the current knowledge does not provide ultimate answer on theantimicrobial mechanism(s) of PEF, it seems that there is reasonabledoubt if the membrane integrity is the only factor to be considered.

Numbers of factors play a role in the determination of efficiency ofPEF technology as a microbial-inactivation process. Among those thekey role can be attributed to the type of the equipment used, setting ofthe treatment parameters, the type of media/food processed, and thetarget microorganism (Aronsson, Ronner, & Borch, 2005; Barbosa-Canovas and Sepulveda, 2005; De Azeredo, De Oliveira, & De Faro, 2008;Jin et al., 2009; Wouters, Bos, & Ueckert, 2001). When cultures of L.monocytogenes were treated with PEF at 50 °C more than 4 log CFU/mlwas obtained in different types of milk (whole milk, 2% and skimmilk),whereas only 1–3 log CFU/ml reductionwasdetermined at 25 °C (Reina,Jin, Zhang, & Yousef, 1998). The greater reduction observed at highertemperature might be due to greater thermal energy delivered to cells,or due to the fact that thermal energy induced damages in L.monocytogenes cells which were more susceptible to PEF treatment(Fleischman, Ravishankar, & Balasubramaniam, 2004). Similar findingswere reported for E. coli O157:H7 and Salmonella Typhimurium DT104,where the greater sensitivity at higher temperature was only related toenhanced thermal energy (Jin et al., 2009; Ravishankar et al., 2002). Theinfluence of pH on the inactivation efficiency of PEF is unclear. Severalreports indicated that Gram-positive L.monocytogenes, Lactobacillus andBacillusweremore resistantwhen treatedwith PEF in citrate-phosphatebuffer with neutral pH thanwith lowpH (Garcia, Gomez, Raso, & Pagan,2005; Saldana et al., 2009;Wouters et al., 2001). Gram-negative bacteriasuch as Salmonella, E. coli, E. coli O157:H7 and Yersinia eneterocoliticashowed more resistance in citrate-phosphate buffers with low pH,compared to neutral (Alvarez, Raso, Palop, & Sala, 2000; Garcia, Gomez,Raso et al., 2005; Jin et al., 2009; Saldana et al., 2009). However,Aronsson and Ronner (2001) found that Gram-negative E. coli showedan opposite behaviour, and more resistance in neutral environment.Recently the study of Jin et al. (2009) suggested that the effect of pH onthe inactivation efficiency of S. typhimurium DT104 was dependent onthe treatment temperature, with cultures beingmore resistant in acidicpH at 25 °C, and more sensitive in acidic pH at 15 °C. Therefore, smallchange in one processing parameter for example temperature, presenceof salt, pH, composition of the treatment medium may have animportant influence on the final inactivation effect by PEF, which canexplain much dissimilarity in published data. Several studies indicatedthat emulsified lipids, soluble proteins or conductive food particulatesdo not have any protective effect against microbial inactivation by PEF(Dutreux et al., 2000; Manas, Barsotti, & Cheftel, 2001), although otherresearchers determined the protective role of fats and proteins (Martin,Qin, Chang, BarbosaCanovas, & Swanson, 1997). The relationshipbetween these factors and their overall contribution to the measuredeffectives of the PEF still requires further investigation.

Like for probably all less-than sterilization treatments the level ofsub-lethal injury of the microbial cells is an important factor that canbe detrimental for the safety, and quality of treated foods. Severalreports have not demonstrated the existence of sub-lethally injuredbacterial subpopulations when cells were exposed to PEF treatment(Wuytack et al., 2003). However, some recently published reportsconfirmed the induction of sub-lethal injury in bacterial culturesthrough PEF treatment (Garcia, Gomez, Condon, Raso, & Pagan, 2003;Garcia, Gomez, Manas et al., 2005; Picart, Dumay, & Cheftel, 2002;Ravishankar, Fleischman, & Balasubramaniam, 2002). E. coli O157:H7,S. Typhimurium, Y. enterocolitica suffered between 90 and 99% sub-lethal injury induced by PEF treatment when suspended in buffer pH4.0 (Garcia, Gomez, Manas et al., 2005). The work of Saldana et al.(2009) showed that E. coli strains were more susceptible to sub-lethalinjury than different strains of L. monocytogenes, S. aureus and S.Typhimuriumwhen subjected to PEF, although a great strain variationwas noticed. Perni, Chalise, Shama, and Kong (2007) also found 99% ofinjured cells when exposing E. coli K12 and S. Typhimurium toelectrical pulses of 32 ns duration at a field intensity of 100 KV/cm and

a repetition rate of 30 pulses per second for a total of 300 s. Whilebehaviour of cultures containing no sub-lethally injured cells is morepredictable (what is dead is dead, andwhat is alive is viable) it is also afact that sub-lethally injured cells can be further suppressed andpossibly killed by an appropriate set of extrinsic and intrinsic factors.

The study of Khadre and Yousef (2002) investigated the effect ofPEF on virus, and determined that rotavirus showed great resistanceagainst PEF treatment that comprised of 20–29 KV/cm, pulse duration3 µs, with total treatment time of 145.6 µs. It seems that PEF is lesseffective against protein capsids found in viruses as compared withlipid-rich membranes in bacteria.

Regarding the efficiency of PEF against bacterial spores, differentresults are reported. Spores of B. cereus showed high PEF resistancewith less than 0.5 log cycles reduction when treatment with 25 KV/cm, 8.3 pulses was applied, and temperature after the treatment wasbetween 23 and 30 °C (Cserhalmi, Vidβcs, Beczner, & Czukor, 2002).However the study of Marquez, Mittal, and Griffiths (1997) reportedmore than 5 log cycles for B. cereus spores suspended in 0.15% NaClsolution when treated with high voltage PEF (50 KV/cm, 50 pulses at25 °C). This disagreement in reported data might be correlated todifferent treatment parameters used, but also the medium in whichspores were resuspended.

PEF-treated food productsmay only be placed on themarket in theEuropean Union (EU), after having examinedwhether they fall withinthe scope of Regulation (EC) No. 258/97 concerning novel foods andnovel food ingredients. Therefore, additional information on thepossible undesired changes which may occur after the interactionbetween the product and PEF process are needed. Food and DrugAdministration (FDA) approved the use of PEF in the preservation ofliquid eggs in 1996 (Dunn, 1996), while the first commercial PEFapplication for fruit juice preservation was installed in the UnitedStates in 2005 (Clark, 2006).

5. Intense light pulses

Intense light pulses (ILP) is one of emerging non-thermaltechnologies investigated as an alternative to the traditional thermaltreatments. It is used to decontaminate surfaces by killing micro-organisms using short time pulses of an intense broad spectrum, richin UV-C light (the portion of the electromagnetic spectrumcorresponding to the band between 200 and 280 nm), which hasbeen proven to be effective for microbial inactivation. Severalsynonyms are used to describe this technology, such as pulsed light(Oms-Oliu, Martin-Belloso, & Soliva-Fortuny, 2010), high intensitybroad spectrum pulsed light (Roberts and Hope, 2003), pulsed whitelight (Dunn, 1996), pulsed UV light (Bialka and Demirci, 2007; Bialka,Demirci, & Puri, 2008) and intense light pulses (Gomez-Lopez,Devlieghere, Bonduelle, & Debevere, 2005).

There are currently very few commercially available ILP systemsfor industrial use. However, it is expected that in number ofcommercial applications ILP will replace continuous UV systems.Possible example is the UV tumbling process that was developed byC&S Equipment Co. Where either a rotating drum or screw conveyorlifts and tumbles the product to ensure exposure to the UV source.Based on our research experience it is not difficult to imagine that ILPcan be integrated in such a system. The unit can be used to treat freshvegetables, fruits, meats, frozen products, and cooked, refrigeratedproducts (Koutchma, Forney, & Moraru, 2009).

The mode of action of the pulsed light process is attributed to theeffect of the high peak power and the UV component of the broadspectrum of the flash. ILP has been successfully used to inactivatebacteria and fungi in foods and fruits and contact surfaces (Anderson,Rowan, MacGregor, Fouracre, & Farish, 2000; Bialka and Demirci,2007; Krishnamurthy, Demirci, & Irudayaraj, 2007; Ozer and Demirci,2006). Several selected studies that reported efficiency of ILP on theinactivation of E. coli O157:H7 and L. monocytogenes are presented in

Table 5The efficiency of intense light pulses against E. coli O157:H7 and L. monocytogenes.

Pathogen Food product Treatment conditions Log reduction Reference

E. coli O157:H7 Agar 3 J/cm2, 200 pulses, 100 ns 6.2 Rowan et al. (1999)Agar 7 J/cm2, 50 pulses, 30 µs 4.7 Gomez-Lopez et al. (2005)Agar 3 J/cm2, 512 pulses, 1 µs 6.8 MacGregor et al. (1998)Salmon fillets 5.6 J/cm2, 180 pulses 1.09 Ozer and Demirci (2006)Alfalfa seeds 5.6 J/cm2, 270 pulses 4.89 Sharma and Demirci (2003)Apple cider 1.05 J/cm2, 12 pulses, 360 µs 3.22 Sauer and Moraru (2009)Apple juice 1.05 J/cm2, 12 pulses, 360 µs 2.52 Sauer and Moraru (2009)Strawberries Total energy dose 64.8 J/cm2, 180 pulses 3.3 Bialka et al. (2008)Raspberries Total energy dose 72 J/cm2, 180 pulses 3.9 Bialka et al. (2008)Blueberries Total energy dose 32.4 J/cm2, 180 pulses 4.9 Bialka and Demirci (2007)

L. monocytogenes Agar 3 J/cm2, 200 pulses, 100 ns 4.4 Rowan et al. (1999)Agar 7 J/cm2, 50 pulses, 30 µs 2.8 Gomez-Lopez et al. (2005)Agar 3 J/cm2, 512 pulses, 1 µs 6.25 MacGregor et al. (1998)Agar 1.5 J/cm2, 1 pulse, 300 µs 1.6 Elmnasser et al. (2007)Salmon fillets 5.6 J/cm2, 180 pulses 1.02 Ozer and Demirci (2006)TSBYEa 7 J/cm2, 20 pulses, 30 µs ≈1.5 Rajkovic et al. (2009a), Van Houteghem et al. (2008)

a TSBYE – Tryptone Soya Broth Yeast Extract.


Table 5. Gomez-Lopez et al. (2005) did not observe any sensitivitypattern among different groups of microorganisms, after studying 27bacterial, yeast and mould species, while decreasing order ofsensitivity was observed by Anderson et al. (2000): Gram-negativebacteria, Gram-positive bacteria and fungal spores. The contradictionin reported data might be due to the experimental setups andtechnology (equipment) used.

The mechanism of microbial inactivation by ILP is mainlyexplained through the photochemical effect, which includes chemicalmodification and cleavage of DNA, protein denaturation and otheralterations of cellular materials, thus preventing cells to replicate(Anderson et al., 2000; Wekhof, 2000). Photothermal and photo-physical effects have been also proposed to explain mechanism ofinactivation during the ILP treatment (Krishnamurthy et al., 2007;Wekhof, 2000; Wuytack et al., 2003).

Wuytack et al. (2003) determined the occurrence of sub-lethalinjury in ILP treated cultures of S. Typhimurium when selective mediasuch as violet red bile glucose agar (VRBG) and Tryptone Soya Agar(TSA) supplemented with 3% NaCl were used, while no sub-lethalinjury occurred when TSA pH 5.5 was used. Sub-lethal injury was alsodetermined for L. inoccua cells inoculated on the stainless-steelsurface coupons treated with different number of intense light pulses(Woodling and Moraru, 2005). However, the studies of Rajkovic et al.(2009b) and Van Houteghem (Van Houteghem et al., 2008) indicatedthat ILP induced the least injury in treated L. monocytogenes, incomparison to the treatments with lactic acid and chlorine dioxide.For this reason no extension in the lag phase of ILP-treated L.monocytogenes under suboptimal conditions was noticed in compar-ison to non-treated cells.

The inactivation efficacy of pulsed light depends on the intensity(measured in J/cm2) and number of pulses delivered (Gomez-Lopez,Ragaert, Debevere, & Devkghere, 2007). Additionally the distancefrom the source of the light and sample, the thickness of the product,the opacity of the liquid samples, presence of particulate materials arecritical parameters that should be optimized to obtain maximaleffectiveness of the treatment (Krishnamurthy et al., 2007). ILP seemsto be less effective for oil and protein rich food, whereas foods rich incarbohydrates seems to be more suitable for this technology (Gomez-Lopez et al., 2005). Gomez-Lopez et al. (2005) described that for anindustrial implementation: the position and orientation of strobes inan unit would determine the lethality, that products to be treatedshould be flashed as soon as possible after contamination occurs, thata cooling system should be used for heat-sensitive products and thatflashed products should be light protected. However, it is important to

note that latest results reported by Rajkovic et al. (2009a) revealedthat repetitive cycles of inactivation with ILP could result in anincreased resistance in L. monocytogenes and E. coli O157:H7.

The effectiveness of ILP against viruses was also reported (Lamontet al., 2007; Roberts and Hope, 2003). When poliovirus andadenovirus were suspended in phosphate buffer solution, 4 and 1log reduction were determined, respectively, using energy of pulsedUV-light of 11.5 mJ/cm2 (Lamont et al., 2007). The same studyindicated that 2.3 J/cm2 were needed to obtain reduction of 4 log foradenovirus, indicating great resistance of this virus against pulsed UV-light. Roberts and Hope (2003) determined >4.8–>7.2 log cycleswhen viruses were treated with energy of 1 J/cm2 of intensity broadspectrum pulsed light. However, the presence of protein in suspend-ing media decreased the efficiency of pulsed light and energy of 2 J/cm2was needed to achieve 5 log reduction. Gomez-Lopez et al. (2005)observed 3.7 and >5.9 level of inactivation for spores of Bacilluscirculans and B. cereus, when used 50 intense light pulses, with 7 J/cm2

per pulse. Spores of Aspergillus niger showed great resistance againstILP, which could be attributed to the dark pigment of the spores. Thiswas confirmed by strong absorbance of A. niger spores in UV region(Anderson et al., 2000), indicating that the absorbance characteristicsmight play an important role in protective mechanism against UVlight pulses.

An important advantage of ILP is that it can be relatively simplyapplied in processing line after the heat treatment (e.g. after slicing ofcooked ham) where possible post-heat treatment contaminationwould be eliminated prior to packaging. Moreover, an application ofILP seems to be legally situated easier than many other moderntechnologies. In the United States, the FDA (Code 21CFR179.41, issuedby the Food and Drug Administration, US FDA, 2005) approved the useof pulsed UV light in the production, processing, and handling of foodand food-contact surfaces for the control of surface microorganisms,provided that the treatment uses a xenon lamp with emission ofwavelengths between 200 and 1000 nm, with a pulse width notexceeding 2 ms, and the cumulative level of the treatment notexceeding 12 J/cm2. In the member states of European Unionregulation EC 258/97 requires proof that new technologies are notsignificantly altering the nutritional value or chemical composition offood. In most cases pulsed light treatment does not modify the treatedproduct, and in that case legal approval is much suppler. However, ananalytical studymust prove this for each new application. Suggestionsmade elsewhere that ILP treated foods need to comply with legalframework designed for radiation-treated foods (Koutchma et al.,2009) does not seem plausible.


6. Weak organic acid

Weak organic acids are frequently used as an inexpensive andeffective intervention to reduce number and prevalence of bacterialpathogens on food products. Of all organic acids evaluated in theliterature, acetic and lactic acid are found to be themost acceptable. Theapplication of 2% lactic acid spray solution on beef carcasses and chickenbreasts has been effective in reducing population of E. coli O157:H7 formore than 1.5 log CFU/cm2 (Anang, Rusul, Bakar, & Ling, 2007;Bosilevac, Nou, Barkocy-Gallagher, Arthur, & Koohmaraie, 2006;Kalchayanand et al., 2008). More than 6 log CFU/cm2 of E. coli O157:H7was reducedwhen 2% lactic acid solutionwas applied on the surfaceof cantaloupes (Materon, 2003). Table 6 summarizes recent studies inwhich lactic acid was used to inactivate E. coliO15:H7, L. monocytogenesand C. jejuni. Several factors should be considered to achieve optimalactivity when organic acids are applied as surface decontaminants, suchas the type of acid, concentration, pH and temperature of the acidsolution, but also the type, pH, buffering capacity of the food product,and the initialmicrobial load.When the increased concentrationof lacticacid solution was used, the greater reduction in cell count wasdetermined (Castillo et al., 2001). As expected the efficiency of organicacid solutions also increased with the decrease of pH in the solution(Van Netten, Huis in 't Veld & Mossel, 1994a).

In general Gram-negative bacteria are more susceptible to decon-taminationwith organic acid thanGram-positive (Corry, James, James,&Hinton, 1995; Van Netten, Huis in 't Veld, & Mossel, 1994c; Virto, Sanz,Alvarez, Condon, & Raso, 2006). Among Gram-negative bacteria, E. coliO157:H7 shows great acid resistance. An opposite, effect of organic acidwas more pronounced in Gram-positive cultures of S. aureus and C.perfringens than Gram-negative E. coli and Salmonella (Raftari, Jalilian,Abdulamir, Sekawi, & Fatimah, 2009; Skrivanova, Marounek, Benda,Brezina, 2006). The authors explained this by the fact that Gram-positive bacteria are more susceptible to the action of compoundsinterfering with the transport of ions across the cell membrane.

The mechanism of inactivation by weak organic acids lays down inthe ability of undissociated form of organic acid to penetrate throughthe cell membrane, and to dissociate inside the cell, resulting indecreased intracellular pH value, which is essential for the control of

Table 6Selected studies evaluating the efficiency of lactic acid on the inactivation of E. coli O157:H


E. coli O157:H7 Chicken breast 2% lactic acid, 10 min dip, at 25 °CChicken breast 2% lactic acid, 30 min dip, at 25 °CBeef head 2% lactic acid, 26 s spray, 25 °CBeef 2% lactic acid, spray, at 42 °CBeef 2% lactic acid, 5 min dip, 23 °CBeef Prechill water wash followed by postchill

4% lactic acid spray, at 55 °C, for 15 sBeef Prechill water wash followed by postchill

4% lactic acid spray, at 65 °C, for 15 sBeef 10% lactic acid, 30 s spray, at 55 °CTomatoes 2% lactic acid, 15 s dip, at 5 °C

Cantaloupes 1.5% lactic acid, 1 min dip, 25 °CTomatoes 0.2% lactic acid, 1 min dip, at 25 °CLettuce 1% lactic acid, 1 min dip, at 25 °C

L. monocytogenes Beef 2% lactic acid, 5 min dip, 23 °CBeef 2% lactic acid, 15 s dip, 24–25 °CBeef 4.5% lactic acid, 2 min dipMung bean sprouts 2% lactic acid, 10 min dip, 22 °CLettuce 1% lactic acid, 90 s dip, 22 °CIceberg lettuce 0.5% lactic acid, 2 min dip, 20 °C

C. jejuni Chicken skin 2.5% lactic acid, 1 min dip, at 25 °CChicken meat 2.5% lactic acid, 1 min dip, at 25 °CSuspension 1% lactic acid, 1 min, at 4 °CChicken carcasses 2% lactic acid, spray, for short timePork belly 2% lactic acid, pH 2.3, 30 s spray, at 21 °C

ATP synthesis, RNA and protein synthesis, DNA replication and cellgrowth (Booth, 1985). Beside the decrease in intracellular pH, theperturbation of the membrane functions by organic acid moleculemight be also responsible for the microbial inactivation. The highconcentration of anions (due to dissociation) inside the cells mightresult in an increased osmolarity and consequently to the metabolicperturbation (Hirshfield, Terzulli, & O'Byrne, 2003).

As for other non-thermal inactivation treatments, the microbialsub-lethal injurymight occur when the decontaminationwith organicacids is applied (Lee, Yun, Fellman, & Kang, 2002; Liao, Shollenberger,& Phillips, 2003). Alexandrou, de, and Adams (1995) reported thatweak organic acids such as acetic and lactic acid showed greaterability to inflict the subpopulation of sub-lethal injured cells thanstronger hydrochloric acid. Lactic acid treatment induced sub-lethalinjury in L. monocytogenes and E. coli O157:H7 cultures inducingprolonged lag phase during subsequent storage in MAP at lowtemperature (Smigic et al., 2009a; Van Houteghem et al., 2008).

Organic acids might be applied as sprays washes or dippingsolutions, depending on the food product to be decontaminated andon the infrastructure of the processing plant. When used as carcassesdecontamination rinses, they should be used in early stages of theproduction, after hide removal, before or after the evisceration, butbefore chilling. The possible discoloration, flavour and/or odour due tothe treatment with organic acids should be evaluated for each of thefood product decontaminated (Smulders and Greer, 1998). Decon-tamination of carcasses with organic acid showed minimal effect onthe sensory quality of meat (Pipek et al., 2005), although somechanges might occur when applied directly on the meat cuts (Theronand Lues, 2007). The risk that might be associated with the use oforganic acid as a decontamination intervention in food industry isrelated to potential induction of acid adaptation (acid habituation oracid tolerance). It is of note that lactic acid resistant mutants of L.monocytogeneswere selected after 20 successive cycles of exposure tolactic acid treatment (Rajkovic et al., 2009a).

Organic acids such as lactic, citric and acetic acids at concentrationof 1.5–2.5% have been already approved as acceptable innervations forreduction of microbial pathogens on meat carcasses in the UnitedStates (FSIS, 1996a). However, European Union only recently

7, L. monocytogenes and C. jejuni.


1.79 Anang et al. (2007)2.59 Anang et al. (2007)1.52 Kalchayanand et al. (2008)1.60 Bosilevac et al. (2006)

≈1 Mustapha, Ariyapitipun, and Clarke (2002)4.2 Castillo et al. (2001)

>4.5 Castillo et al. (2001)

3.4 Carlson et al. (2008)≈3 Ibarra-Sanchez, Alvarado-Casillas, Rodriguez-Garcia,

Martinez-Gonzales, and Castillo (2004)6.60 Materon (2003)2.2 Velazquez, Barbini, Escudero, Estrada, and Guzman (2009)1.7 Velazquez et al. (2009)1.56 Ariyapitipun, Mustapha, and Clarke (2000)1.09 Ozdemir et al. (2006)

≈2 Palumbo and Williams (1994)2 Lee et al. (2002)

<1 Samara and Koutsoumanis (2009)2.8 Akbas and Olmez (2007)1.69 Riedel, Bronsted, Rosenquist, Haxgart, and Christensen (2009)

≈0.8 Riedel et al. (2009)≈0.5 Zhao and Doyle (2006)≈0.7 Bolder, Lipman, and Putirulan (2004)

2.5 Van Netten, Huis in 't Veld, and Mossel (1994b)


provided the legal bases to permit the use of substances other thanpotable, clean water to decontaminate products of animal origin (EU,2004). Nevertheless, their use is still not authorized in EU as the use oflactic acid in meat processing plants was not positively evaluated bythe European Food Safety Authority (EFSA) penal due to insufficientdocumentation (EFSA, 2006).

7. Aqueous chlorine dioxide

Chlorine dioxide (ClO2) is a powerful oxidising and sanitizingagent, with a broad biocidal activity against bacteria, yeast, viruses,fungi and protozoa. ClO2 is yellow-greenish gas, which is highlysoluble in water, but, unlike chlorine, ClO2 does not react with water.The oxidation capacity of ClO2 is determined by its oxidation numberof chlorine atom which is +4, and it can accept 5 electrons whencompletely reduced to chloride ion. As opposite to chlorine whichreacts via oxidation and electrophilic substitution, ClO2 only reacts byoxidation, therefore it does not produce, or to a very limited extend,organochlorine compounds (Richardson et al., 2000; WHO, 2000).

ClO2 might be used for the disinfection of drinking water, fordecontamination of fresh produce (Gomez-Lopez, Rajkovic, Ragaert,Smigic, & Devlieghere, 2009; Gomez-Lopez et al., 2008; Wu and Kim,2007), but also in fish, meat and poultry processing (Andrews, Key,Martin, Grodner, & Park, 2002; Emswiler, Kotula, & Rough, 1976; Kim,Marshall, Du, Otwell, & Wei, 1999). The efficiency of ClO2 seems to bedependent on the food product which is decontaminated, but also onthe concentration of ClO2 and duration of the treatment. Cutter andDorsa (1995) used ClO2 spray washes for beef decontamination inorder to evaluate influence of ClO2 concentration, contact time andapplication pressure. They found that ClO2 reduced aerobic platecounts present on beef by<1 log CFU/cm2 at low pressure, and at highpressures with longer contact times, reductions increased to 0.93–1.52 CFU/cm2. These results were not significantly different fromwater controls. From this study, the researchers concluded that ClO2 isineffective as a decontaminant of beef carcasses against bacteria offaecal origin (Cutter and Dorsa, 1995; Emswiler et al., 1976). L.monocytogenes and C. jejuni inoculated on the chicken meat werereduced by 0.63–1.93 log CFU/g when exposed to 100 ppm ClO2 for10 min (Hong, Ku, Kim, & Song, 2007). Recently the study performed

Table 7Selected studies evaluating the efficiency of liquid ClO2 on the inactivation of E. coli O157:H

Pathogen Food product Treatment conditio

E. coli O157:H7 Lettuce 10 ppm ClO2 liquidLettuce 20 ppm ClO2 liquidIceberg lettuce 20 ppm ClO2 liquidIceberg lettuce 200 ppm ClO2 liquiBaby carrot 20 ppm ClO2 liquidFish 100 ppm ClO2 ice,Apples 5 ppm ClO2 liquid,Spinach 100 ppm ClO2 liquiLettuce 5 ppm ClO2 liquid,Alfalfa seeds 25 ppm ClO2 liquid

L. monocytogenes Whole apples 3 ppm liquid ClO2,Sliced apples 3 ppm liquid ClO2,Whole lettuce 3 ppm liquid ClO2,Shredded lettuce 3 ppm liquid ClO2,Strawberries 3 ppm liquid ClO2,Cantaloupes 3 ppm liquid ClO2,Uninjured surfaces of green pepper 0.3 ppm liquid ClOBlueberries 15 ppm liquid ClO2

Chicken breast 100 ppm ClO2 liquiChicken legs 100 ppm ClO2 liqui

C. jejuni Chicken breast 50 ppm ClO2 liquidChicken legs 50 ppm ClO2 liquidChicken breast 100 ppm ClO2 liquiChicken legs 100 ppm ClO2 liquiChicken carcases 4.25 ppm ClO2 liqu

on shrimp and crawfish indicated total reduction of aerobic andphychrotropic plate count when 40 ppm ClO2 was used (Andrewset al., 2002). However, higher levels of reduction by liquid ClO2 weredetermined in the studies when decontaminating fruits and vege-tables, as it can be seen from the results presented in Table 7.Additionally, the efficiency of ClO2 is highly dependent on the state ofthe product, as it was noticed that wounds or injuries on the surface ofthe products significantly decreased the antimicrobial activity of thisdecontamination agent (Han, Linton, Nielsen, & Nelson, 2001).

The exact mechanism of microbial inactivation by ClO2 is notcompletely understood. Although early work by Bernarde, Snow,Olivieri, and Davidson (1967) performed on E. coli indicated that thelethal lesion produced by ClO2 to microbial cells is directly related toprotein synthesis. Loss of permeability control was identified later byBerg, Roberts, and Matin (1986) as the primary lethal event at thephysiological level of ClO2 on bacterial cells. The effect of ClO2 wasrelated to non-specific oxidative damage of the outer membraneleading to the destruction of the trans-membrane ionic gradient. Thedenaturation of constituent proteins critical to cellular integrity andfunction, through the covalent oxidative modification of tryptophanand tyrosine residues, are also found to be implicated in the lethalevent of ClO2 (Ogata, 2007). Young and Setlow (2003) proposed thatsome type of membrane damage could be the cause of B. subtilisspores death, since ClO2 treated spores can undergo the initial steps inspore germination, but cannot go further in this process. B. cereus cellstreated with ClO2 exhibited surface roughness and indentations, andwere elongated, in contrast with control cells, which were uniformrods with smooth surfaces (Peta, Lindsay, Brozel, & von Holy, 2003).Elongation of cells might result from the inhibition of division andassociated metabolic damage.

Regarding effectiveness of ClO2 against viruses, greater inactiva-tion was determined in alkaline than in neutral pH of suspendingmedium (Alvarez and Obrien, 1982). It is of note that most of thereported data were obtained in studies of water disinfection, whiledecontamination of viruses in food has been less evaluated.

The application of liquid ClO2 in the processing of fresh produce iseasily applied to the existing process during washing withoutmodifying subsequent steps. For decontamination of carcasses inpoultry processing, ClO2 might be applied either as sprays or washes.

7, L. monocytogenes and C. jejuni.

ns Log reduction Reference

, 5 min 1.2 Singh, Singh, Bhunia, and Stroshine (2002), 15 min 1.7 Singh, Singh, and Bhunia (2003), 2 min 0.83 Keskinen, Burke, and Annous (2009)d, 2 min 1.43 Keskinen et al. (2009), 15 min 2.5 Singh et al. (2003)120 min 4.8 Shin, Chang, and Kang (2004)10 min ≈1.5 Huang et al. (2006)d, 5 min ≈2.0 Lee and Baek (2008)10 min ≈1.5 Huang et al. (2006), 5 min ≈1.0 Singh et al. (2003)5 min >5 Rodgers, Cash, Siddiq, and Ryser (2004)5 min 4.6 Rodgers et al. (2004)5 min >5 Rodgers et al. (2004)5 min 4.6 Rodgers et al. (2004)5 min >5 Rodgers et al. (2004)5 min >5 Rodgers et al. (2004)2, 10 min 1.87 Han et al. (2001), 2 h 4.88 Wu and Kim (2007)d, 10 min 0.63 Hong et al. (2007)d, 10 min 1.93 Hong et al. (2007), 10 min ≈1.0 Hong et al. (2007), 10 min 0.1 Hong et al. (2007)d, 10 min 1.21 Hong et al. (2007)d, 10 min 0.99 Hong et al. (2007)id ≈0.7 Bolder et al. (2004)


It might be also added into the chiller water to avoid potentialmicrobial cross-contamination. Application of ClO2 for the surfacedecontamination of fresh produce or meat carcasses is not legalized inEU. Although the directive EC 853/2004 (EU, 2004) states that foodbusiness operators can use substance, other than potable water, forsurface decontamination, this is applicable only if the substance hasbeen approved by Scientific panel of European Commission (EFSA,2005). In the United States food processors are legally allowed to useClO2 as disinfectant (FDA, 2008; FSIS, 1996b). Amaximum 3 ppm ClO2

concentration is allowable in water that is in contact with fresh fruitsand vegetables or in chilled poultry water and 200 ppm is allowablefor sanitizing processing equipment.

8. Food preservation by combined processes (hurdle technology)

Majority of treatments that do not cause complete inactivation ofmicroorganisms induce sub-lethal injury to the present bacterial cells.Depending on the type of the injury, type of the organism andsurrounding environment these injured bacterial cultures have thepotential to resuscitate and resumegrowth under favourable conditions(Smigic et al., 2009a; Van Houteghem et al., 2008). In addition to theinactivation technologies applied to foods, both microbial growth andsurvival can be influenced by different intrinsic factors of the food. Thisfurthermeans that intrinsic factors (water activity, pH, nutrients), aloneor combined with the extrinsic factors (modified atmosphere, temper-ature, humidity), can enhance or inhibit recovery and growth of micro-bial cells. Therefore, the safety and stability of food can be improvedusing an appropriate combination of several factors that will preventsurviving and proliferation of sub-lethally injured cells. These multipleintrinsic factors are part of a dynamic system that changes from themoment of application to the moment of consumption. During thisprocess, each factor plays a role of a different magnitude and suchmagnitude changes over time (Raso et al., 2005 and references therein).

Food preservation by combined processes (hurdle technology)supports the combination of existing and novel preservation techni-ques to establish a series of factors (hurdles) that microorganism (ofconcern) should not be able to overcome (Leistner and Gorris, 1995;Raso and Barbosa-Canovas, 2003; Raso et al., 2005). This is especiallyimportant for the microorganisms and bacterial spores that are veryresistant to applied inactivation technologies, and are a limiting factorfor the application of mild treatments. To apply principles of foodpreservationby combinedprocesses correctly, an understanding of themechanisms of action of the individual factors alone and incombination is needed. This understanding allows justified and wellbalanced combination of hurdles to achieve desired level of safety andquality, avoiding the need to apply only one factor at such highintensity that causes severe changes in the quality of food (Raso et al.,2005). Instead, using combined hurdles one can interfere with themicrobial homeostasis and extend the effect of sub-lethal injury. Thismay not only result in growth inhibition, but can also impair survivalpossibilities leading to death of injured microbial cells.

As mentioned in this review, mild decontamination technologiesinflict the subpopulation of sub-lethally injured cells. Van Houteghem etal. (2008) studied the effects of carbon dioxide inmodified atmosphereson the resuscitation of L. monocytogenes cells injured by ILP, ClO2, lacticacid and mild heat treatments during storage at 7 °C. The resultsindicated additional bactericidal effects of CO2 on cultures treated withlactic acid, liquid ClO2 and ILP, with additional reductions in viable L.monocytogenes of 0.5–1.0 log CFU/ml. Lag phase duration was signifi-cantly different between the different treatments, with non-treated cellsshowing the shortest lag phase, followed by that of heat, ILP, lactic acidand finally ClO2 treated cells. The authors have found relationship be-tween the amount of sub-lethally damaged cells after amild inactivationtreatment and the lag phase duration in the CO2 environment. Similarly,Rajkovic et al. (2009b) reported on the effect of partial inactivation of L.monocytogenes with lactic acid, liquid ClO2 and ILP on the injury and

post-treatment growth behaviour under increased NaCl concentrationand reduced pH values. The results showed that the inactivation levelsand the percentage of sub-lethal injured cells were dependent uponstrain and type of inactivation technique used. The greatest effect on thegrowth retardation was at every pH observed for the cultures treatedwith ClO2, followed by lactic acid and ILP. Under increased NaCl con-centration lactic acid treated cells suffered hardest growth retardation,followed by ClO2 and ILP, respectively. Recovery of ILP treated cultureswas not always different from untreated cultures. In general, damagedmicroorganisms becomemore exacting in growth requirements and aremore sensitive to other preservation factors like low pH, antimicrobialcomponents, etc. (Raso et al., 2005).

In foods preserved by combined methods the microbial homeo-stasis is threatened on different multiple sites asking for a complexand energy demanding microbial response (Raso et al., 2005). Thisfact enables the obtaining of safe and stable foods by balancingdifferent factors and strategies. Particularly under mildly lethalstress, the ultimate cause of inactivation is subject of cellularresponse to additional regulation that integrates information aboutthe global state of the cell and its environment (Aertsen andMichiels, 2004). It is therefore an art of combining differentsuboptimal factors that will push microbial cell over the thin linebetween bacterial growth and inactivation. The extended post-treatment effect based on the growth retardation or inhibition ofinjured cells under sub-optimal conditions can be utilized as animportant tool in conditioning of microbial food safety.

9. The risks to be considered

The food industry aims increasingly at applying novel and mildpreservation techniques for the production of food products that willmeet demands of a modern consumer. As seen in examples describedabove in the principles of food preservation by combined processes forthe production of mildly preserved food products, an inactivation stepis often applied as a first step. Most often, a mild heat treatment isapplied for this purpose. However, the industry resortsmore andmoreto non-thermal alternatives such as high hydrostatic pressure,decontamination with organic acids or other chemical agents (e.g.chlorine dioxide) and intense light pulses. When these types of noveland mild inactivation technologies are applied, incomplete inactiva-tion and sub-lethal damage of the target microorganisms is oftenobtained, but by the application of suboptimal intrinsic and extrinsicfactors of food, possible growth during shelf-life of food productmightbe prevented or further inactivation promoted. Acknowledgement ofthe resistance of spores tomild inactivation treatments resulted in twostep inactivation strategies where one step has a task to inducegermination of present spores, and the second delayed step has a taskto inactivate newly formed vegetative cells. However, the hiddendanger lays in incomplete germination and thus incomplete inactiva-tion of present cells. This can endanger both microbial quality andmicrobial safety of treated foods.

In the scientific community, but also among food processors andlegislators there is a concern about the fact that the application of sub-lethal stress factors could induce (cross) resistance mechanisms in thesurviving population and change their virulence characteristics (Hill,Cotter, Sleator, & Gahan, 2002; Lou and Yousef, 1997; Rajkovic et al.,2009a; Rowan, 1999). This increased resistance stems from the fact thatbacteria, as living organisms, can respond to and harness themselvesagainst stresses to which they are exposed. Increased resistance hasbeen recently documented as a consequence of repetitive treatments ofmild inactivation. The occurrence thereof was found to be the one sidespecies and even strain dependent, and on the other side treatmentdependent. In none of the cases the increase was abrupt; rather it wasobserved as a steady decrease in the original sensitivity towards appliedtreatment (Rajkovic et al., 2009a). The physiological and molecularresponses by which certain food-borne bacterial pathogens adapt,


acquire resistance andmodify their virulence patterns to these complexinimical stresses are multifarious (Archer, 1996). For instance, Rowanet al. (1999) summarizes elsewhere published data on external factors,which influence on the expression of virulence factors has beendocumented, e.g. CO2 (B. anthracis and Vibrio cholera), temperature(Bordetealla pertussis, E. coli, Salmonella Typhimurium, L.monocytogenes,V. cholerae, Shigella and Yersinia species), pH (Salmonella Typhimurium,E. coli, L. monocytogenes, V. cholerae), osmolarity (Pseudomonasaeruginosa, L. monocytogenes), oxidative stress (S. Typhimurium) etc.The complexity of the phenomena and its practical importance to publichealth requires further qualification and quantification of theseresponses. Moreover, the understanding the molecular and cellularmechanisms of these adaptive responses is of great interest, because itmay lead to improved strategies for combating microbes, not only infoods, but also in disease.

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Contemporary strategies in combating microbial contamination in food chain

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