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Review

Non-proteolytic

Clostridium

botulinum spores in

low-acid cold-

distributed foods and

design of

pasteurization

processes

Filipa V.M. Silvaa,b,* andPaul A. Gibbsc

aDepartment of Chemical and Materials

and Engineering, University of Auckland,

Private Bag 92019, Auckland 1142, New Zealand

(Tel.: D 64 9 3737999; fax: D 64 9 3737463;

e-mails: filipa.silva@auckland.ac.nz;

filipavinagresilva@gmail.com)bInstituto Nacional dos Recursos Biologicos (INRB),

Fonte Boa, 2005-048 Vale de Santarem, PortugalcLeatherhead Food International, Randalls Road,

Leatherhead, Surrey KT22 7RY, UK

(e-mail: pgibbs@leatherheadfood.com)

The increasing consumption of minimally processed chilled

foods poses new risks in terms of public safety and foodborne

infections. Thermal pasteurization (65e95 �C) reduces the

numbers of unwanted vegetative cells of pathogenic and spoil-

age microorganisms in foods, extending food shelf-life, pro-

moting food safety, and allowing the reduction and

elimination of added chemical preservatives to foods. With

respect to low-acid foods, microbial spores surviving pasteur-

ization must be controlled, by using cold storage and transpor-

tation (1e8 �C), and a limited shelf-life. A review on the heat

resistance of most common contaminant microbial flora in

low-acid chilled foods is presented and a new strategy for

the design of pasteurization processes based on the non-pro-

teolytic psychrotrophic Clostridium botulinum spores thermal

resistance will be approached.

Introduction to thermal pasteurization of foodsThe global incidence of foodborne disease is difficult to

estimate. However, 1.8 million people were reported tohave died in 2005 from diarrhoeal diseases. A high propor-tion of these cases can be attributed to contamination offood and in particular drinking water (WHO, 2007). Low-acid foods have been the cause of human diseases suchas gastroenteritis and listeriosis. Common symptoms offoodborne illness include diarrhoea, stomach cramps, fever,headache, vomiting, dehydration and exhaustion. Propercooking or thermal processing of foods can eliminatemost of the causative agents of foodborne diseases. Ther-mal pasteurization is a traditional physical process offood decontamination which is still in common use todayand will be used in future, for being efficient, environmen-tally friendly, healthy and inexpensive when compared withother preservation technologies. The mild temperaturesused allow greater retention of the original properties ofthe raw food. In low-acid chilled foods, the main goal ofpasteurization is the reduction of pathogens responsiblefor foodborne illness and human disease (Fig. 1), whereasin the case of high-acid foods, pasteurization is intendedto avoid spoilage and economic losses. This work presentsa review focused on the heat resistance of relevant microbesin pasteurized and chilled foods with low acidity (pH> 4.6)completed with a section with recommendations for thedesign of pasteurization processes based on non-proteolyticClostridium botulinum. Although microbial spoilage ofthermally processed foods can be caused by incipient spoil-age (growth of bacteria before processing) or re-contamina-tion after processing (leakage), we will focus our attentionon the survival and growth of thermoresistant microorgan-isms (e.g. spore-formers) due to insufficient heat processing.

Classical and modern concepts of food pasteurizationThe word ‘pasteurization’ has its origin in the work of

the French scientist Louis Pasteur, and refers to a mild* Corresponding author.

0924-2244/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2009.10.011

Trends in Food Science & Technology 21 (2010) 95e105

Author's personal copy

heat treatment (50e90 �C) used for food preservation,which aims to inactivate vegetative forms of pathogenicand spoilage microorganisms. Unlike sterilization, afterpasteurization the food is not sterile since heat-resistant mi-crobial spores are present (Lund, 1975, Chap. 3) (Fig. 1).Therefore, other forms of preservation such as refrigeration(e.g. milk), atmosphere modification (e.g. vacuum packag-ing, meats, cheeses), addition of antimicrobial preservatives(e.g. common salt e sodium chloride, sodium nitrite, ascor-bic acid, sorbic acid, sulphur dioxide, etc.), or combinationsof the referred techniques, are required for product stabili-zation and distribution. Exceptions are some processedfoods that possess constituents or ingredients that are anti-microbial under certain conditions, and not allowing micro-bial growth: e.g. fermented foods containing alcohol or acid(e.g. wine, beer, pickles), carbonated drinks (e.g. sodas),very sweet foods presenting aw< 0.65 or SS> 70 �brix(e.g. honey, jams, jellies, dried fruits, fruit concentrates),or salty foods (e.g. salted fish or meats). Other exceptionsinclude the high-acid foods (pH< 4.6), which are stableat ambient conditions after a pasteurization process, be-cause the acidic food environment is not conducive to thegrowth of harmful microorganisms and microbial sporesin the pasteurized food. For these types of foods(pH< 4.6), a pasteurization process allows a long shelf-life (months) at room temperature (Ramaswamy & Abbate-marco, 1996), and if refrigerated storage is used, a milderpasteurization may be applied and the product quality isimproved (Fig. 1). However, regarding low-acid food prod-ucts (pH> 4.6, e.g. milk), a shorter shelf-life (days) is ob-tained after pasteurization, and refrigerated storage isnecessary to maintain product safety during storage, by

restricting the growth of surviving pathogens (e.g. spore-formers) in the food (Adams & Moss, 1995; Fellows,1988; Potter, 1986).

Pasteurization was recently re-defined by the UnitedStates Department of Agriculture as: ‘‘any process, treat-ment, or combination thereof, that is applied to food to re-duce the most resistant microorganism(s) of public healthsignificance to a level that is not likely to present a publichealth risk under normal conditions of distribution and stor-age’’ (NACMCF, 2006). This definition, therefore, includesnon-thermal pasteurization processes such as high pressure(HP) and high intensity pulsed electric fields, and the ef-fects of these new technologies on microorganisms andthe foods are an essential research line (Hendrickx &Knorr, 2001; Hulsheger, Potel, & Niemann, 1981;Lehmann, 1996). Nevertheless, the efficacy of HP in termsof spore (Lee et al., 2006) and spoilage endogenous enzymeinactivation is limited (Raso & Barbosa-Canovas, 2003;Van Buggenhout, Messagie, Van der Plancken, & Hen-drickx, 2006). After studying combinations of heat andpressure for the destruction of C. botulinum spores, Mar-gosch et al. (2006) expressed concerns since higher sporesurvival was observed when using temperature and highpressure treatments simultaneously, in comparison to theexclusive use of temperature.

Low-acid foods requiring a cold distribution chainMinimally heated chill-stored foods have been increas-

ing by 10% each year in market volume, since they are con-venient (ready-to-eat, with longer shelf-life than fresh) andcan better retain the original properties of the foods. Asmentioned previously, and for reasons of public safety,low-acid pasteurized foods are stored, transported andsold under refrigerated conditions and with a limitedshelf-life (Fig. 1), to minimize the outgrowth of pathogenicmicrobes in the foods during distribution. Beverages suchas milk, dairy products (e.g. cheeses), poultry/meat/fish/vegetable products (e.g. cooked cured ham, hot-smokedfish, soup), some shellfish (e.g. cockles), some sauces,food ingredients, low carbonated drinks, and certain fruitjuices (e.g. pear, some tropical juices) are examples oflow-acid pasteurized foods. Refrigerated processed foodsof extended durability (REPFED) are also included in thisclass. These are generally packaged under vacuum ‘sous-vide’ or modified atmospheres to ensure anaerobic condi-tions and submitted to mild heat treatments, being storedfrom a few days to several weeks depending on the foodand severity of the heat process.

Equations for process design and assessmentAlthough deviations from linearity (e.g. shoulder, tail,

sigmoidal-like curves, biphasic curves, concave and convexcurves) have been reported and remain unexplained, in par-ticular with vegetative pathogens such as Escherichia coli,Salmonella spp., Listeria monocytogenes, Staphylococcusaureus (Juneja, Snyder, & Marmer, 1997; Valdramidis,

Fig. 1. Pasteurization of foods.

96 F.V.M. Silva, P.A. Gibbs / Trends in Food Science & Technology 21 (2010) 95e105

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Geeraerd, Bernaerts, & Van Impe, 2006), most of the liter-ature available in this area assumes as linear the log of sur-vivors vs. time of inactivation (Eqs. (1) and (2)).Fortunately, the inactivation of the most heat-resistantform of microorganisms, the spore, generally follows thefirst-order/Bigelow model pattern (Bigelow & Esty, 1920;Silva, Gibbs, Vieira, & Silva, 1999; Silva & Gibbs, 2009;Teixeira, 1992):

N

No

¼ 10�t

DT ð1Þ

DT ¼ DTref� 10

�Tref �T

z

�ð2Þ

where: N e number of microbial spores, N0 e initial num-ber of microbial spores, DT e decimal reduction time (min)at temperature T, t e time (min), DTref e decimal reductiontime at a reference temperature and z e number of degreesCelsius required to reduce D by a factor of 10.

During the pasteurization process, the integrated lethal-ity at a single point within the food container, also knownas pasteurization value (P), is the equivalent time of pas-teurization at a certain temperature (Tref) expressed in min-utes (Eq. (3)) (Shapton, 1966):

P¼Z PT

0

10

�T�Tref

Z

�dt ð3Þ

where: P¼ pasteurization value (min), PT¼ total processtime (min), Tref¼ reference temperature for the pasteuriza-tion target and z¼ z-value for the pasteurization target.

Heat resistance of microbes targeting the productionof safe and stable low-acid chilled foods (pH > 4.6)

Before discussing microbial targets of pasteurization inlow-acid cold-stored foods, we must know that the sporeis the most heat-resistant microbial form, a highly resistantdehydrated form of a dormant cell produced under condi-tions of environmental stress and as a result of ‘‘quorumsensing’’. Moulds, certain yeasts and bacteria can producespores, although mould spores are not as heat resistant asbacterial spores. Heat is the most efficient method for sporeinactivation, and is presently the basis of a huge worldwideindustry (Bigelow & Esty, 1920; Gould, 2006). Microbialspores are much more resistant to heat in comparison totheir vegetative counterparts, generally being able to sur-vive the pasteurization process. Spore heat resistance mayalso be affected by the food environment in which the or-ganism is heated (Tables 1 and 2). For instance, spores(and vegetative cells) become more heat resistant at lowwater activity (Harnulv & Snygg, 1972; Murrel & Scott,1966; Silva et al., 1999). If, after pasteurization, the storagetemperature as well as the food characteristics (pH, wateractivity, food constituents) are favourable for sufficienttime, surviving spores can germinate and grow to attainhigh numbers (e.g. 107/g or mL) and cause foodborne

diseases or spoilage. Control of spores during storage ofpasteurized foods requires an understanding of both theirheat resistance and outgrowth characteristics. This sectionstarts with a brief description of pathogenic microbes of rel-evance, followed by a review of the heat resistance of ther-mally driven microbiological criteria to be considered astargets in designing pasteurization processes for low-acidrefrigerated foods. Thermal resistances are listed fromhigher to lower D- and z-values.

Pathogens of concern in low-acid chilled foodsVarious pathogens can be associated with foodborne dis-

eases and outbreaks from improperly processed/preserved/stored low-acid chilled foods. With respect to public health,the most dangerous spore-formers in low-acid chilled foodsare the psychrotrophic non-proteolytic strains of C. botuli-num (Carlin, Girardina et al., 2000; Gould, 1999). In spiteof the low incidence of this intoxication, the mortality rateis high, if not treated immediately and properly. Thesestrains of C. botulinum have been implicated in human bot-ulism incidents from ingestion of the following contami-nated foods (Lindstrom, Kiviniemi, & Korkeala, 2006):hot-smoked fish (Pace, Krumbiegel, Angelotti, & Wisniew-ski, 1967), canned tuna fish in oil (Mongiardo et al., 1985),canned truffle cream/canned asparagus (Therre, 1999), pas-teurized vegetables in oil (Aureli, Fenicia, & Franciosa,1999), canned fish (Przybylska, 2003), and canned eggplant(Peredkov, 2004). Bacillus cereus is another spore-formingand pathogenic bacteria detected in pasteurized and chilledfoods such as cooked rice and other chilled foods contain-ing vegetables (Carlin, Girardina et al., 2000; Carlin,Guinebretiere et al., 2000), since some strains of B. cereuscan grow at low temperatures (T< 8 �C) (Choma et al.,2000; Dufrenne, Bijwaard, te-Giffel, Beumer, & Noter-mans, 1995; Dufrenne, Soentoro, Tatini, Day, & Noter-mans, 1994; Garcıa-Armesto & Sutherland, 1997). Thereare some non-pathogenic spore-formers including Bacillusand Clostridium spp. (Broda, Saul, Lawson, Bell, & Mus-grave, 2000), and moulds that can cause significant eco-nomic losses to food producers. For example, Bacilluscirculans was identified as the major spoilage Bacillus incommercial vegetable purees pasteurized and stored at4 �C (Carlin, Guinebretiere et al., 2000). Very limiteddata on spoilage and thermal resistance of spore-formersis available in the literature.

Other examples of foodborne infections from raw andheated foods include L. monocytogenes (milk, soft cheese,ice cream, cold-smoked fish, chilled processed meat prod-ucts such cooked poultry), E. coli serotype O157:H7 (vero-toxigenic E. coli VTEC; beef, cooked hamburgers, raw fruitjuice, lettuce, game meat, cheese curd), Salmonella enteri-tidis (poultry and eggs), Vibrio parahaemolyticus (improp-erly cooked, or cooked, re-contaminated fish and shellfish),Vibrio cholerae (water, ice, raw or under-processed sea-food), and foodborne trematodes from fish/seafood pro-duced by aquaculture (FDA, 1992; Keiser & Utzinger,

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2005; WHO, 2002). Pasteurized milk and dairy productsmay also be contaminated with Brucella, thermophilicStreptococcus spp., and Mycobacterium avium paratubercu-losis (MAP) (Grant, 2003; Grant, Ball, Neill, & Rowe,

1996; Westhoff, 1978), which can be infectious at lowcell numbers, although they cannot grow at chilltemperatures. Coxiella burnetii, the causative agent of‘Q-fever’, can also be a problem in milk (Cerf & Condron,

Table 1. Heat resistance of psychrotrophic (group II) non-proteolytic strains of Clostridium botulinum spores.

Food product Spore inoculum, botulinum strains T (�C) D-value (min) z-value (�C) T range (�C) Reference

Crabmeat Mixture of threestrains: ‘Ham’, ‘Kapchunka’,17B

88.9 13 8.6 88.9e94.4 Peterson et al. (1997)90.6 8.292.2 5.394.4 2.9

Codhomogenate

ATCC 25765 (type B) 75.0 54 8.6 75.0e92.0 Gaze and Brown (1990)80.0 1885.0 4.090.0 1.1

ATCC 9564 (type E) 75.0 59 8.3 75.0e92.080.0 1585.0 4.890.0 0.79

Turkey slurry KAP B5 75.0 33 9.4 75.0e90.0 Juneja et al. (1995)90.0 0.80

Carrothomogenate

ATCC 25765 (type B) 75.0 19 9.8 75.0e92.0 Gaze and Brown (1990)80.0 4.285.0 1.690.0 0.43

ATCC 9564 (typeE) 75.0 18 9.8 75.0e90.080.0 4.385.0 0.7390.0 0.48

Turkey slurry ‘Alaska’ 70.0 52 9.9 70.0e85.0 Juneja et al. (1995)85.0 1.2

Whitefish paste ‘Alaska’, ‘Beluga’, 8E,‘Iwanai’, ‘Tenno’

80.0 1.6e4.3 5.7e7.6 73.9e85.0 Crisley, Peeler, Angelotti, and Hall (1968)

Blue crab ‘Alaska’, ‘Beluga’, crab G21-5,crab 25V-1, crab 25V-2

73.9 6.8e13 7.0e8.4 73.9e85.0 Lynt, Solomon, Lilly, and Kautter (1977)76.6 2.4e4.179.4 1.1e1.782.2 0.49e0.74

Oysterhomogenate

‘Minnesota’, ‘Alaska’,G21-5, 25V-1, 25V-2

73.9 2.0e9.0 4.2e7.1 73.9e82.2 Chai and Liang (1992)82.2 0.080e0.43

Table 2. Heat resistance of Clostridium perfringens and Bacillus cereus in low-acid foods (pH > 4.6).

Bacteria Spore inoculum Food product T (�C) D-value (min) z-value (�C) T range (�C) Reference

C. perfringens 3 strains: NCTC 8238/9,ATCC 10288

Ground turkey 99 23 nr nr Juneja and Marmer (1996)

3 strains: DSM 11784 &NCTC 10614 (incidents),NCTC 08237

Pork luncheon roll 90 31 8.3 90e100 Byrne et al. (2006)95 9.7

100 1.93 strains: NCTC 8238/9,ATCC 10288

Marinated chickenbreast

90 14 nr nr Juneja, Fan, Pena-Ramos,Diaz-Cinco, andPacheco-Aguilar (2006)

B. cereus 3 strains: DSM 4313 (incident),DSM 626, NCTC 07464

Pork luncheon roll 85 30 8.6 85e95 Byrne et al. (2006)90 1095 2.0

Psychrotrophic strainINRA AVTZ415

Distilled water 85 16 8.2 85e100 Fernandez et al. (2001)90 3.995 1.0

100 0.24

nr e not reported.

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2006). Psychrotrophic spoilage microbes such lactic acidbacteria (LAB) (Lactobacillus spp., Leuconostoc spp., Car-nobacterium spp.), moulds (Thamnidium spp., Penicilliumspp.) and yeasts (Zygosaccharomyces spp.), can occur inchilled low-acid foods during storage, in general due topost-process contamination. These are very heat sensitive,for example LAB D63 �C is 14 s in meat sausages (Franz& vonHoly, 1996) and D60 �C is 33 s in milk (De-Angeliset al., 2004).

Psychrotrophic strains of C. botulinumC. botulinum is an anaerobic mesophilic microorganism

(26e37 �C), and the strains causing human botulism belongto groups I (proteolytic, neurotoxins types A, B and F) and II(non-proteolytic, neurotoxin types B, E and F). Strains ofgroup I cannot grow below 10 �C and are not a concern incold-distributed foods. On the contrary, the spores of thenon-proteolytic strains are psychrotrophic, being able to ger-minate and grow at temperatures as low as 3 �C (Schimdtet al., 1961). Under almost any conditions where growth oc-curs, there will be toxin production, being necessary a popu-lation of ca. 105 cells/ml to generate enough toxin to killa mouse. Additionally, outbreaks in chill-stored foods notpasteurized (e.g. fermented seal flipper, fermented salmoneggs, low salt cold-smoked fish), are evidence that thesespores do not require a heat shock to be activated and germi-nate. Psychrotrophic strains of C. botulinum although moreheat sensitive (Table 1) than proteolytic Group I strains, areable to survive to mild heat treatments such as pasteuriza-tion, and, therefore, refrigerated storage conditions are re-quired (Peck, 2006). Cold storage of these foods canreduce or at least retard toxin production, given that this or-ganism needs much longer storage periods to produce the le-thal toxin: e.g. within 31 days at 3.3 �C in beef stew(Schmidt, Lechowich, & Folinazzo, 1961); within 22 daysat 8.0 �C (Betts & Gaze, 1995); �55 days at 4.4 �C, 8days at 10 �C and 2 days at 24 �C in crabmeat homogenates(Cockey & Tatro, 1974). A study carried out with six strainsof psychrotrophic non-proteolytic type E C. botulinum inmilk, could not detect growth during 22 weeks storage at4.4 �C, but strains ‘Beluga’, ‘Tenno’, VH and ‘Alaska’ couldgrow and produce toxin at 10 �C after 21, 28, 42 and 56days, respectively, while at 7.2 �C only ‘Tenno’ could pro-duce toxin after 70 days (Read, Bradshaw, & Francis,1970). Thus, to control human botulism in low-acid pasteur-ized foods the use of refrigerated storage (T< 8 �C) is re-quired with a restricted shelf-life (ACMSF, 1992; Gould,1999). Additional measures of safety with this risky classof foods include the use of added preservatives such salt(>3.5%) and nitrites (>100 ppm) (e.g. cured meat products)(Graham, Mason, & Peck, 1996). The unique use of suchlevels of salts are not sufficient to inhibit the proteolyticstrains of C. botulinum (Group I strains mentioned previ-ously) in pasteurized meat products, and these must be re-frigerated (<8 �C).

The mild pasteurization process applied to REPFED fol-lowed by extended storage at chill temperatures, favours thesurvival and growth of anaerobic spore-forming psychrotro-phic C. botulinum (Lindstrom et al., 2006). Concern with thismicrobe is also extended to semi-preserved foods such cured,cooked ham, cold-smoked fish, fermented marine foods anddried fish (Peck, 2006). Psychrotrophic strains of C. botuli-num present different thermal resistances depending firston the heating menstrum (the food), and in some cases onthe strain. Similar results were obtained with non-proteolyticstrains producing toxin types E and B using cod homogenateand carrot homogenate as heating media (Gaze & Brown,1990; Table 1). Thermal resistance data obtained from the lit-erature (Table 1) revealed that D90 �C varies from seconds tomore than 8 min, D85 �C from a few seconds to 37 min, andD80 �C from a few minutes to 140 min. Crabmeat presentedthe highest D-value, 2.9 min at 94.4 �C, using a mixture of‘Ham’, ‘Kapchunka’ and 17B botulinum strains (Petersonet al., 1997). Most of the authors published similar z-values,ranging between 7 �C and 10 �C. Oyster homogenate pre-sented the lowest heat resistance for the five spore strainscocktail (two from outbreaks in ‘Alaska’ and ‘Minnesota’)of C. botulinum studied, the D- and z-values being the lowestrecorded (Chai & Liang, 1992).

Other pathogenic spore-forming bacteriaTable 2 shows thermal resistance data of the spore-forming

pathogens Clostridium perfringens and B. cereus, which havebeen responsible for outbreaks in low-acid under-pasteurizedchilled foods. Studies with spores of six strains of C. perfrin-gens demonstrated no growth at T� 10 �C, although sporegermination and extended survival at low temperatures oc-curred (De-Jong, Rombouts, & Beumer, 2004). However,knowing that C. perfringens exhibits one of the fastest growthrates of food-associated bacteria, doubling in numbers every8 min at 44 �C, being the most common foodborne illnesscaused by a spore-former, it was also considered in this review(Table 2). As a result of temperature abuse during distributionor storage of heated/cooked foods (e.g. meat products), C. per-fringens may grow, especially in establishments where largequantities of foods are prepared several hours before serving(e.g. school cafeterias, hospitals, nursing homes, prisons,etc.) with concomitant difficulties in rapid chilling to below10 �C. Spores of C. perfringens are more heat resistant thanthose of non-proteolytic C. botulinum, for example in turkeyD99 �C¼ 23 min, D90 �C¼ 31 min in pork roll andD90 �C¼ 14 min in chicken breast.

Eleven strains of isolated psychrotrophic strains of B.cereus able to grow at �7 �C in foods, presented a 2.2 min -<D90 �C< 9.2 min in phosphate buffer (Dufrenne et al.,1995). Other published data gave D90 �C of 10 min and4 min in pork roll (Byrne, Dunne, & Bolton, 2006) and wa-ter (Fernandez, Ocio, Fernandez, & Martınez, 2001), re-spectively. Psychrotrophic strains of B. cereus seem to bemore heat resistant than psychrotrophic strains of C. botu-linum (Tables 1 and 2, Fig. 2).

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Non-spore-forming psychrotrophic pathogensThe pathogenic vegetative bacteria L. monocytogenes,

Yersinia enterocolitica and V. parahaemolyticus, are alsoable to grow in foods with pH> 4.6 at temperatures lowerthan 6 �C (Penfield & Campbell, 1990) but they are easilyeliminated with a few seconds at 70 �C or less (Table 3).Other non-spore-forming pathogens such as E. coli, somestrains of Salmonella, and Aeromonas hydrophila (George,2000; Palumbo, Call, Schultz, & Williams, 1995; Papageor-giou, Melas, Abrahim, & Angelidis, 2006; Schoeni, Glass,McDermott, & Wong, 1995) can also be a problem inchilled foods. However, pasteurization of a few secondsat 65 �C or less should be sufficient to destroy these micro-organisms (Table 3). In general, a few seconds at 65 �Cwere enough to achieve one decimal reduction in Listeria,E. coli, Salmonella, and a few seconds at 60 �C were nec-essary for Y. enterocolitica populations. A. hydrophila pre-sented the lowest heat resistance.

The potential for the growth of other vegetative bacterialpathogens such as Campylobacter spp., V. cholerae, Shi-gella spp., S. aureus, Enterococcus spp. (FDA, 1992; Jay,2000, Chap. 3) in pasteurized/chilled foods is very lowsince apart from being heat sensitive, food must be temper-ature abused during distribution to allow their growth.

Chilled milk is a particular low-acid food of high con-sumption. Although the effect of Mycobacterium aviumparatuberculosis (MAP) in humans is not known yet, thisbacterium causes disease in cattle and as a precaution thedesign of pasteurization in milk and dairy products shouldconsider the thermal resistance of this bacterium. D-valuesin milk at 63 �C, 66 �C and 72 �C are 15 s, 5.9 s and<2.03 s, respectively, and z-value is 8.6 �C (Pearce,Truong, Crawford, Yates, & Cavaignac Sde Lisle, 2001),although Keswani and Frank (1998) obtained much higherD-values in milk (D63 �C 1.6e2.5 min). C. burnetii in milkhas D-values of 4.14 min at 62.8 �C and 2.21 s at 71.7 �C,z-value¼ 4.34 �C (Cerf & Condron, 2006). The high tem-perature short time (HTST) pasteurization standardwhereby milk is held at 71.7 �C for at least 15 s (or anequivalent process such as 62.7 �C for 30 min), was de-signed to achieve a 5-log reduction in the number of viable

microorganisms in milk (European Economic Community,1992; Stabel & Lambertz, 2004). Pasteurizations of 72 �Ce 15 s, 75 �C e 20 s and 78 �C e 25 s resulted in 4 to>6 log reduction in MAP (McDonald, O’Riley, Schroen,& Condron, 2005).

Use psychrotrophic C. botulinum as reference for thedesign of pasteurization in low-acid refrigerated foods

As opposed to the design of sterilization processeswhere a ‘‘botulinum cook’’ must be applied (12D on pro-teolytic C. botulinum, 3 min at 121 �C/250�F) to allow am-bient storage of any food, the criteria for the design ofpasteurization processes varies with the food product, andin low-acid foods, refrigerated storage is a necessary further‘hurdle’ to overcome the reduced severity of the heat treat-ment and assure food safety. The food industry designs pas-teurization processes many times based on experience witha particular food product and has the responsibility to pro-duce a safe food.

General considerations to set a new pasteurizationprocess

Before one can design a pasteurization process for a newfood product, the following must be known or specified: (i)if food is to be stored or distributed at ambient or if a coldchain is necessary; (ii) the desired shelf-life; (iii) if con-sumers belong to susceptible groups (e.g. babies, elderly,hospitalized).

Food products in general can have a shelf-life of �10days as long as they achieve at least one of the following:water activity aw< 0.97, aqueous salt >3.5%, pH< 5.0,heat treatment of 90 �C/10 min (FSA, 2008). However, iffood producers wish to have a food with extended shelf-life or reduced level of chemical preservatives, the follow-ing information must be gathered to set an appropriate pas-teurization process:

(1) identify the microbes (pathogens and spoilage) includ-ing spores able to grow in the food to be pasteurized,capable of causing food degradation, under the storageconditions (e.g. temperature, atmosphere);

(2) obtain from the literature heat resistance data (D- and z-values), regarding those particular microbes able togrow in the food to be pasteurized;

(3) select the most dangerous/thermal resistant microbe asthe pasteurization target;

(4) always check for C. botulinum growth potential in thenew low-acid food;

(5) experimentally determine D- and z-values of target mi-croorganism in the new food;

(6) set a minimum P (pasteurization value) which deliversat least 6D on the most heat-resistant microbe in thefood slowest heating point (Betts & Gaze, 1992);

(7) perform food challenge/storage tests to validate exper-imentally the thermal process under specific storageconditions (T� 10 �C, for low-acid foods);

Psychrotrophic microbes: D-value of seconds at 55°C 60°C 65°C 70°C 75°C 80°C 85°C 90°C 95°C >95°C

Listeria monocytogenes Bacilluscereus

Escherichia coli O157:H7 Clostridium botulinum Salmonella spp.

Yersinia enterocolitica

Aeromonas hydrophila

Mycobacterium avium

Coxiella burnetii

Lactic acid

bacteria Yeasts

Fig. 2. D-values in the range of seconds within the pasteurization tem-peratures shown ( e spore-forming pathogens; e non-spore-form-

ing pathogens; e non-spore-forming spoilage microbes).

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Table 3. Heat resistance of non-spore-forming pathogenic microbes in low-acid foods (pH > 4.6).

Bacteria Food product T (�C) D-value (min) z-value (�C) T range (�C) Reference

Listeriamonocytogenes

Ground pork 55.0 47 5.9 55.0e70.0 Murphy, Beard, Martin, Duncan, and Marcy(2004)70.0 0.085

Chicken gravy 50.0 119e195 5.2e6.1 50.0e65.0 Huang, Yousef, Marth, and Matthews(1992)65.0 0.19e0.48

Cooked lobster 51.6 97 5.0 51.6e62.7 Buduamoako, Toora, Walton, Ablett, and Smith(1992)54.4 55

57.2 8.360.0 2.462.7 1.1

Rainbow trout roe 60.0 1.6 5.4 60.0e Miettinen, Arvola, and Wirtanen (2005)63.0 0.44 63.0

Liquid egg yolk 60.0 1.3 6.1 60.0e62.2 Schuman and Sheldon (1997)62.2 0.58

Liquid egg white 55.1 7.6 9.4 55.1e58.358.3 3.5

Vacuum-packedminced beef

50.0 36 4.2 50.0e60.0 Bolton et al. (2000)55.0 3.260.0 0.15

Escherichiacoli O157:H7

Ground Pork 55.0 33 4.9 55.0e70.0 Murphy, Beard et al. (2004)70.0 0.048

Fully cooked frank 55.0 25 5.1 55.0e70 Murphy, Davidson, and Marcy (2004)70.0 0.038

Raw frank 55.0 2170.0 0.031

Ground beef 55.0 21 6.0 55.0e65.0 Juneja et al. (1997); Juneja and Marmer(1999)65.0 0.39

Ground meats: lamb,chicken, turkey, pork

55.0 11e12 6.5e6.8 55.0e65.065.0 0.29e0.38

Ground morcilla sausage 54.0 5.5 7.4 54.0e62.0 Oteiza, Giannuzzi, and Califano (2003)58.0 2.162.0 0.60

Ground meat:beef (7e20% fat),pork sausage (7e30% fat),chicken (3e11% fat),turkey (3e11% fat)

50.0 50e115 4.4e4.8 50.0e60.0 Ahmed, Conner, and Huffman (1995)55.0 6.4e1960.0 0.37e0.58

Salmonellaspp.

Ground pork 55.0 46 5.9 55.0e70.0 Murphy, Beard et al. (2004)70.0 0.083

Green pea soup 60.0 10 5.7 60.0e71.1 Thomas, White, and Longree (1966)65.6 1.0

Thigh meat 55.0 12 6.9 55.0e62.5 Juneja (2007)60.0 3.262.5 0.84

Chicken breast meat 55.0 6.1 8.1 55.0e62.560.0 3.062.5 0.66

Liquid egg yolk 60.0 0.28 3.5e4.3 55.0e62.2 Schuman and Sheldon (1997)62.2 0.087

Liquid egg white 55.1 8.058.3 1.0

Yersiniaenterocolitica

Vacuum-packedminced beef

50.0 21 nr 50.0e60.0 Bolton et al. (2000)55.0 1.160.0 0.55

Whole and skim milks 62.8 0.17e0.18 nr nr Toora, Buduamoako, Ablett, and Smith(1992)

Aeromonashydrophila

Liquid whole egg 48.0 3.6e9.4 5.0e5.6 48.0e60.0 Schuman, Sheldon, and Foegeding(1997)60.0 0.026e0.040

nr e not reported.

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(8) use preservatives for additional safety or longer shelf-life.

Regarding the low-acid foods, cold-distributed and refer-ring to point 1, all the microbes mentioned in the previoussection can potentially grow at pH> 4.6 and low tempera-tures. Two useful tools for assessing microbial growth po-tential in a specific food environment are freely availablethrough the internet, the ComBase Browser (http://www.combase.cc) and the Pathogen Modelling Program Online(http://pmp.arserrc.gov/PMPOnline.aspx). The first isa combined database of bacterial growth responses todifferent conditions of temperature, pH, water activity andNaCl content, and the second includes the effect ofsodium nitrite (0e150 ppm) in the microbial growth models.

The resume of the heat resistance literature review pre-sented in Tables 1e3 and Fig. 2 can be very helpful forpoints 2 and 3. Spore-forming psycrotrophic, pathogenicbacteria can be inactivated with temperatures of 90e95 �C, whereas temperatures of 65e70 �C are enough fordestruction of vegetative forms of bacterial pathogens,and even lower temperatures of 55e60 �C are enough fordestruction of psychrotrophic spoilage microbes such pseu-domonas, lactic acid bacteria, moulds and yeasts (Fig. 2).

As mentioned in point 4, with respect to low-acidcooked and chilled foods, food processors have to demon-strate that the processed food is safe, not being capable ofsupporting the growth and toxin production by non-proteo-lytic C. botulinum within the specified temperature rangeand storage life of the food.

Regarding points 5 and 6, it is expected from the pub-lished literature, that a P of 6D would require up to6 min, at temperatures from 55 �C to more than 95 �C(Fig. 2), depending on the pair microbial specie andstrain-food heated.

Referring to point 7, knowing that pasteurization aimsfor partial destruction of a certain microorganism, it is es-sential to confirm experimentally if the thermal processspecified is sufficient to stabilize a certain product duringthe intended storage period.

Knowing that adding synthetic preservatives to foodsshould be avoided (point 8), natural or less harmful addi-tives could be used, in the cases where the thermal processis insufficient for obtaining a safe product.

Non-proteolytic psychrotrophic C. botulinumBased on the toxicity of non-proteolytic C. botulinum for

humans and its higher values of heat resistance compared toother psychrotrophic pathogens, we recommend the use ofthis microbe’s thermal resistance properties as the referenceor indicator for the design of pasteurization processes re-garding low-acid chilled foods.

Current recommendation for low-acid refrigerated foodsis a process (P) of 10 min at 90 �C or equivalent (ACMSF,1992; Gould, 1999; Peck, 2006). As mentioned previously,if this process is not sufficient to achieve 6D inactivation of

C. botulinum, other hurdles such as the addition of preser-vatives are required for safety assurance (ACMSF, 1992). Itis known from the literature that a salt content �3.5% in thefood prevents botulinum growth during chill storage (Peck,2006). The addition of salt to levels of 2.5% and 4.3% to ananaerobic meat medium stored at temperatures from 5 to16 �C, increased the number of days required for thegrowth of non-proteolytic C. botulinum strains (Grahamet al., 1996). Thus, besides refrigerated storage(T< 8 �C), Clostridium and Bacillus spores possibly sur-viving to the pasteurization, must be controlled with salts(>3.5% salt-on-water, e.g. NaCl, sodium lactate) and ni-trites (>100 ppm, e.g. NaNO2).

In certain foods, milder thermal treatments than the rec-ommended 10 min at 90 �C are sufficient for botulinalsafety. For example, crabmeat homogenate submitted to1 min at 85 �C was sufficient to inactivate 106 cfu/g of C.botulinum type E spores (6D) and keep the food safe(non-toxic) for 6 months at 4.4 �C (Cockey & Tatro,1974). Peck, Lund, Fairbairn, Kaspersson, and Undeland(1995) registered botulinum growth in a meat medium pas-teurized at 80 �C for 23 min after 12e40 days storage at 6e12 �C, and recommended a minimum pasteurization of19 min at 85 �C, followed by storage at T< 12 �C anda shelf-life of not more than 28 days to reduce the risk offood botulism. The increase of 5 �C in the pasteurizationtemperature retarded the growth at 6e12 �C storage from12e40 days to within 42e53 days. An even more intensepasteurization at 95 �C for 15 min, did not allow growthat T� 12 �C for 60 days (Peck et al., 1995).

With respect to the variability of D-values determinedfor different C. botulinum strains-foods (Table 1), a mini-mum process of 6 min at the temperatures of 85 �C (oysterhomogenate, blue crab), 90 �C (whitefish paste, turkeyslurry, carrot homogenate), 95 �C (cod homogenate),>95 �C (crabmeat) would be necessary to achieve a P inthe magnitude of 6D.

The range of pasteurization temperatures targetingC. botulinum spores would overcome the problem of crea-tion of resistant ‘‘shock proteins’’ and damaged cells,which otherwise could have a negative impact in terms offood safety.

Future trendsThe move away from adding chemical preservatives to

foods would lead to an increase in the use of healthier tech-niques of food decontamination such as pasteurization(thermal or non-thermal).

Attention should be given to ‘‘recipes’’ for the design ofpasteurization processes and the production of effectivelypasteurized foods, so that outbreaks and foodborne infec-tions can be avoided. The opinion of an expert in thisarea is strongly recommended.

The cold distribution chain (T< 8 �C) should be care-fully monitored and checked for temperature abuses. Thefood package should present visible information regarding

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refrigerated storage to the end consumer. Time-temperatureindicators may be used as well. Increases in temperaturewould potentiate the growth of pathogen/spoilage psychro-trophic microbes and also the survivors of mesophilic (op-timum growth at 10e50 �C) and thermophilic (optimumgrowth at 50e70 �C) spores, including some strains of C.botulinum (Juneja & Marks, 1999) and B. cereus, C. per-fringens, Bacillus coagulans, Bacillus stearothermophilus,Desulfotomaculum nigrificans, and Alicyclobacillusacidoterrestris.

The use of psychrotrophic C. botulinum thermal resis-tance properties for the design of pasteurizations in low-acid foods cold-stored/distributed is suggested.

Careful attention should be given to the microbial inac-tivation results obtained with real commercial processes,which deal with non-isothermal heating.

The conclusions and recommendations for the design ofthermal pasteurisations can be extended to the design ofnon-thermal pasteurization processes, it being necessaryto determine microbial D- and z-values with respect tothe new technologies.

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