食品包装技术（英文版）：36756_12

12.1 Introduction Modified atmosphere packaged (MAP) prepared fresh produce provides substrates and environmental conditions conducive to the survival and growth of microorganisms. Minimal processing treatments such as peeling and slicing disrupt surface tissues, expose cytoplasm and provide a potentially richer source of nutrients than intact produce (Brackett, 1994; Barry-Ryan and O’Beirne, 1998, 2000). This, combined with high Aw and either close to neutral (vegetables) or low acid (many fruits) tissue pH, facilitate microbial growth (Beuchat, 1996). These products can harbour large and diverse populations of microorganisms, and counts of 10 5 –10 7 CFU/g are frequently present. Most bacteria present are Gram-negative rods, predominantly Pseudomonas, Enterobacter or Erwinia species (Brocklehurst et al., 1987; Garg et al., 1990; Magnuson et al., 1990; Manvell and Ackland, 1986; Marchetti et al., 1992; Nguyen-the and Prunier, 1989). The organisms present and counts are affected by product type and storage conditions. Lactic acid bacteria have been detected in mixed salads and grated carrots, and may predominate in salads when held at abuse (30oC) temperatures (Manvell and Ackland, 1986). Yeasts commonly isolated include Cryptococcus, Rhodotorula, and Candida (Brackett, 1994). Webb and Mundt (1978) surveyed 14 different vegetables for moulds. The most commonly isolated genera were Aureobasidium, Fusarium, Mucor, Phoma, Rhizopus, and Penicillium. A number of important human pathogens can also be found in MAP prepared produce. Their presence is a consequence of contamination during agricultural production (mainly from contaminated seed, soil, irrigation water, and air), 12 Reducing pathogen risks in MAP-prepared produce D. O’Beirne and G. A. Francis, University of Limerick, Ireland during harvesting and manual preparation (human contact) or during machine processing and packaging (contaminated work surfaces/packaging materials/ equipment). Cross-contamination by end-users after pack opening can also occur. By extending shelf-life and protecting product quality, MAP prepared produce systems can provide sufficient time for pathogens to grow to significant numbers on otherwise acceptable fresh foods (Berrang et al., 1989b). The risk of food poisoning is greatest in products eaten raw without any further preparation. While the food safety record of these products is good, a comprehensive understanding of the implications of this technology for pathogen survival and growth is required in order to optimise production systems and to inform HACCP protocols. The effects of MAP technology on the survival and growth of non-pathogens and on the interaction between pathogens and non-pathogens is also important (Francis and O’Beirne, 1998b). Non-pathogens are both potential competitors of pathogens and important indicators of product spoilage. While considerable progress has been made in the past decade in our understanding of the safety of these novel and complex food systems there are still significant gaps in knowledge requiring further research. 12.2 Measuring pathogen risks A range of pathogens have been isolated from raw produce (Brackett, 1999; Francis et al., 1999) and foodborne infections have been linked to the consumption of raw vegetables and fruits (Tables 12.1 and 12.2). While pathogens have also been isolated from MAP prepared produce (see Table 12.1) relatively few foodborne infections have been directly linked with this range of products. Those that have been linked include an outbreak of botulism ultimately linked to an MAP dry coleslaw product (Solomon et al., 1990) and a Salmonella Newport outbreak linked to ready-to-eat salad vegetables (PHLS, 2001). There was also an outbreak of shigellosis linked to shredded lettuce (Davis et al., 1988) though exactly how this product was packaged is unclear. Increasing consumption of fresh produce in the United States has been paralleled by an increase in produce-linked food poisoning outbreaks (NACMCF, 1999). Contributory factors include the increased range and diversity of products available to consumers and the elimination of seasonality by almost year-round availability of many commodities. This diversity and availability has been achieved by increased globalisation of the produce trade, and has brought with it new food safety risks and challenges. While the main pathogens of concern are still non-proteolytic Clostridium botulinum, Listeria monocytogenes, Yersinia entercolitica and Aeromonas hydrophila, there are important emerging threats from viral and protozoan pathogens. There are a number of difficulties in estimating the magnitude of the true microbial risk from fresh produce and MAP fresh produce. Studies where samples of produce are examined for the presence of pathogens are, of necessity, 232 Novel food packaging techniques limited in size and may not accurately reflect global contamination levels. In addition, surveys showing the absence of pathogens may receive less attention than those showing their presence, and this may distort the true picture. A recent examination of 127 fresh produce items from the Washington DC area (Thunberg et al., 2002) showed low levels of contamination, no Salmonella or Campylobacter contamination, and seven samples positive for L. monocytogenes. On the other hand, food poisoning incidents related to fresh produce may be under-reported. By comparison with those linked to meat and poultry, outbreaks related to produce do not have the same pathogen and product characteristics which assist in recognition, investigation, and reporting (NACMCF, 1999). For example, the short shelf-lives, complex distribution and universal consumption of fresh produce make produce-implicated outbreaks more difficult to pin down. Even when produce is almost certainly implicated, the exact point of contamination is difficult to prove beyond doubt. Of 27 examples of produce-linked food poisoning outbreaks considered by NACMCF, investigators had definitively identified the point of contamination in only two. The main pathogens of concern in MAP produce are discussed below, focusing on sources and levels of contamination, and their likely health risk to consumers. 12.2.1 Listeria monocytogenes L. monocytogenes is a Gram-positive rod which causes several diseases in man including meningitis, septicaemia, still-births and abortions (ICMSF, 1996). It is considered ubiquitous in the environment, being isolated from soil, faeces, sewage, silage, manure, water, mud, hay, animal feeds, dust, birds, animals and man (Al-Ghazali and Al-Azawi, 1990; Gunasena et al., 1995; Gray and Killinger, 1966; Nguyen-the and Carlin, 1994; Welshimer, 1968). Contamination of vegetables by L. monocytogenes may occur through agricultural practices, such as irrigation with polluted water or use of contaminated manure (Nguyen-the and Carlin, 1994; Geldreich and Bordner, 1971). It may also occur during processing (see Section 12.3.3). L. monocytogenes has been isolated from minimally processed vegetables at rates ranging from 0% (Farber et al., 1989; Fenlon et al., 1996; Gohil et al., 1995; Petran et al., 1988) to 44% (Arumugaswamy et al., 1994; Beckers et al., 1989; Doris and Seah, 1995; Harvey and Gilmour, 1993; MacGowan et al., 1994; McLauchlin and Gilbert, 1990; Sizmur and Walker, 1988; Velani and Roberts, 1991). In France (Nguyen-the and Carlin, 1994) and Germany (Lund, 1993) levels of >10 2 CFU/g are unacceptable, while in the UK and USA the organism must be absent in 25g. Of particular concern is the organism’s ability to grow at refrigeration temperatures; the minimum temperature for growth is reported to be 0.4oC (Walker and Stringer, 1987). It is also facultatively anaerobic, capable of survival/growth under the low O 2 concentrations within MA packages of prepared vegetables. While counts generally remain constant at 4oC (Farber et al., 1998), they can increase to high numbers at mild abuse temperatures (8oC), Reducing pathogen risks in MAP-prepared produce 233 Table 12.1 Occurrence of pathogens on minimally processed produce Vegetable Number (and %) Country and Reference of positive samples comments Listeria monocytogenes Cucumber slices 4/5 (80%) Malaysia Arumugaswamy et al., 1994 Bean-sprouts 6/7 (85%) Malaysia Arumugaswamy et al., 1994 Coleslaw 2/92 (2.2%) Canada Schlech et al., 1983 2/50 (4%) Singapore Doris and Seah, 1995 3/39 (7.7%) United Kingdom MacGowan et al., 1994 Harvey and Gilmour, 1993 Pre-packed mixed salads 3/21 (14.3%) Northern Ireland 4/60 (6.7%) United Kingdom Sizmur and Walker, 1988 Chopped lettuce 5/39 (13%) Canada Odumeru et al., 1997 Cut and packaged lettuce 3/120 (2.5%) Australia Szabo et al., 2000 Prepared mixed vegetables 8/42 (19%) United Kingdom Velani and Roberts, 1991 (contamination during processing suspected; <200/g present) Fresh cut salad vegetables 11/25 (44%) The Netherlands Beckers et al., 1989 (<10 2 /g present) Chicory salads (8.8%) France Nguyen-the and Carlin, 1994 (<1/g present) Prepared vegetables 1/26 (3.8%) United Kingdom MacGowan et al., 1994 Processed vegetables and salads (13%) United Kingdom McLaughlin and Gilbert, 1990 Aeromonas spp. Cut lettuce 66/120 (55%) Australia Szabo et al., 2000 Salad mix 12/12 (100%) Italy Marchetti et al., 1992 Prepared salads (21.6%) UK Fricker and Tompsett, 1989 E. coli O157:H7 Salad mix 0/63 (0%) US Lin et al., 1996 Clostridium botulinum MAP Salad mix 2/350 (0.6%) US Lilly et al., 1996 MAP cabbage 1/337 (0.3) US Lilly et al., 1996 MAP green pepper 1/201 (0.5%) US Lilly et al., 1996 Salmonella spp. Salad mix 1/159 (0.6%) Egypt Saddik et al., 1985 Endive 2/26 (7.7%) Netherlands Tamminga et al., 1978 Yersinia spp. Cut and packaged lettuce 71/120 (59%) Australia Szabo et al., 2000 Prepacked salads 3/3 (100%) UK Brocklehurst et al., 1987 Campylobacter jejuni Mushrooms 3/200 (1.5%) United States Doyle and Schoeni, 1986 particularly after anti-microbial dipping treatments or within nitrogen flushed packages (Francis and O’Beirne, 1997). However, evidence is emerging that levels of virulence may vary greatly among L. monocytogenes strains, and that some serotypes found in MAP produce may be different (and less virulent) than those isolated in food poisoning outbreaks (Beuchat and Ryu, 1997). Further research is required to determine the significance of different L. monocytogenes strains for human health. 12.2.2 Clostridium botulinum Cl. botulinum is a member of the genus Clostridium, characterised as Gram- positive, rod-shaped, endospore forming, obligate anaerobes (Varnum and Evans, 1991). The foodborne Clostridia have been comprehensively reviewed by McClane (1997) and Dodds and Austin (1997). Cl. botulinum is divided into numerous sub-divisions, based on the serological specificity of the neurotoxin produced, and physiological differences between strains. Human botulism is normally attributed to sub-species antigenic types A, B, E and occasionally type F. Endospores of Cl. botulinum are ubiquitous, being distributed in soils, aquatic sediments and the digestive tract of animals and birds. Vegetables are potentially contaminated during growth, harvesting and processing (Rhodehamel, 1992). Despite their ubiquity, a recent study identified only 0.36% of pre-cut MAP vegetables to be contaminated with Cl. botulinum spores (Lilly et al., 1996). In the case of mushrooms, a much lower incidence of Cl. botulinum was reported (Notermans et al., 1989) than had been reported previously (Hauschild et al., 1978), a change attributed to hygienic improve- ments in growing techniques. The possibility of growth and toxin production by Cl. botulinum before obvious spoilage has long been of concern in over-wrapped mushrooms (Sugiyama and Yang, 1975) and in vacuum packaged prepared potatoes (O’Beirne and Ballantyne,1987). In addition, sufficiently anoxic conditions are frequently observed in MA packages where the respiration rate of the product is not matched by the permeability of the packaging used. Anoxic conditions may also develop within MAP produce where edible coatings are used (Guilbert et al., 1996). Highly permeable or perforated over-wrapping films have been used for fresh mushrooms and low storage temperatures and short shelf-lives have been requirements in prepared potato products (IFST, 1990). In the case of other items of vacuum packaged/MAP prepared produce, the data suggest that spoilage is likely to preceed toxin production (Larson et al., 1997; Petran et al., 1995), with a probability of 1 in 10 5 for toxin production to occur prior to obvious spoilage (Larson et al., 1997). However, there is a report linking a botulism outbreak with coleslaw prepared from a MAP dry coleslaw mix (Solomon et al., 1990). The short shelf-lives of retail packs and the good control of temperature/modest storage lives of catering packs are likely to minimise such risks, but there is need for vigilance and further research. 236 Novel food packaging techniques 12.2.3 Escherichia coli O157:H7 E. coli, type species of the type Enterobacteriaceae genus, Escherichia, is a common inhabitant of the gastrointestinal tract of mammals. Despite the commensal status of the majority of strains, pathogenic strains, particularly enterohaemorrhagic E. coli O157:H7, have emerged as highly significant foodborne pathogens. Gastroenteritis and haemorrhagic colitis are classical symptoms, while complications including thrombocytopenic purpura and haemolytic uraemic syndrome have been documented (Martin et al., 1986), the latter potentially leading to renal failure and death in 3–5% of juvenile cases (Karmali et al., 1983; Griffin and Tauxe, 1991). The principal reservoir of E. coli O157:H7 is believed to be the bovine gastrointestinal tract (Wells et al., 1991; Doyle et al., 1997). Hence, contamination of meat and other food products with faeces is a significant risk factor. Contamination of, and survival of the organism in natural water sources make these also potential sources in the distribution of infection, particularly if untreated water is used to wash produce. The potential for cross- contamination during distribution and domestic storage are also of concern. Information regarding contamination rates of MAP prepared vegetables is limited. Recent surveys in the UK and US failed to find this pathogen (FDA, 2001). 12.2.4 Aeromonas hydrophila Aeromonas hydrophila is a motile, Gram-negative, rod-shaped bacterium in the family Vibrionaceae. It causes a broad spectrum of infections (septicaemia, meningitis, endocarditis) in humans, often in immunocompromised hosts, and Aeromonas spp. have been associated epidemiologically with travellers diarrhoea. Its significance as a human pathogen has been reviewed by Altwegg and Geiss (1989). A. hydrophila is considered to be ubiquitous and has been isolated from many sources. The best known sources are treated and untreated water, and animals associated with water, such as fish and shellfish (ICMSF, 1996). Hazen et al. (1978) isolated A. hydrophila from the vast majority of aquatic environments. A. hydrophila is also associated with soil (Brandi et al., 1996) and with a range of foods including fresh vegetables. Foods in which A. hydrophila was isolated were most likely contaminated by water, soil or animal faeces. A. hydrophila possesses a number of characteristics of concern in relation to MAP prepared vegetables. It is a psychrotroph; it grows slowly at 0oC, but temperatures of 4–5oC will support growth in foods. It is also a facultative anaerobe, capable of growing in atmospheres containing low concentrations of oxygen. Marchetti et al. (1992) isolated high counts (10 3 –10 6 /g) of A. hydrophila in commercial MAP prepared vegetable salads. Aeromonas spp. were also recovered from green salad, coleslaw (Hudson and De Lacy, 1991), pre-made salad samples (Fricker and Tompsett, 1989), mayonnaise salad samples (Kn?chel and Jeppesen, 1990) and commercial mixed vegetable salads Reducing pathogen risks in MAP-prepared produce 237 (Garc?′a-Gimeno et al., 1996). By contrast, none of the vegetable samples from shops in Sweden was positive for Aeromonas spp. (Krovacek et al., 1992). 12.2.5 Salmonella Salmonella, a genus of the family Enterobacteriaceae, are characterised as Gram-negative, rod-shaped bacteria. Pathogenic species include S. Typhimurium, S. Enteritidis, S. Heidelberg, S. Saint-paul, and S. Montevideo. Salmonellae are mesophiles, with optimum temperatures for growth of 35–43oC. The growth rate is substantially reduced at <15oC, while the growth of most salmonellae is prevented at <7oC. Salmonella are facultatively anaerobic, capable of survival in low O 2 atmospheres. These organisms are abundant in faecal material, sewage and sewage-polluted water; consequently they may contaminate soil and crops with which they come into contact. Sewage sludge may contain high numbers of salmonellae and, if used for agricultural purposes, will disseminate the bacterium. Once introduced into the environment, salmonellae remain viable for months (ICMSF, 1996). Potential contamination from workers who handle produce in the field or in processing plants is of great concern (see Section 12.4). Salmonellae have not generally been found in MAP produce, though they have been isolated from bean-sprouts (20%) in Malaysia (Arumugaswamy et al., 1994). 12.2.6 Yersinia enterocolitica Y. enterocolitica is currently considered to be the most significant genus member with respect to foodborne disease (Varnum and Evans, 1991). Traditional gastrointestinal symptoms, potentially mediated through the activity of a heat-stable enterotoxin, may develop into suppurative and autoimmune complications (Robins-Browne, 1997). The psychrotrophic status of Y. enterocolitica is potentially of great significance with regard to refrigerated MAP prepared produce. Y. enterocolitica occupies a broad range of ecosystems including the intestinal tract, birds, flies, fish and a variety of terrestrial and aquatic ecosystems. However, most environmental isolates lack virulence markers and are of doubtful significance for human or animal health (Delmas and Vidon, 1985). Isolation of Yersinia spp. from raw vegetables has been reported at rates ranging from 3.3% (Tassinari et al., 1994) to 46.1% (Delmas and Vidon, 1985), although specific isolation rates of pathogenic Y. enterocolitica strains are likely to be significantly lower. 12.2.7 Campylobacter jejuni Since their principal identification as human gastrointestinal pathogens in the 1970s (Butzler et al., 1973; Skirrow, 1977) members of the thermophilic campylobacters, e.g. C. jejuni, have emerged as major human gastrointestinal 238 Novel food packaging techniques pathogens (Ketley, 1997). Despite fastidious growth requirements, members of the genus survive at refrigeration temperatures for extended periods within nutrient limited environments. This property, combined with the low infective dose (Robinson, 1981) and their microaerophilic nature, indicates the potential significance of the genus with respect to refrigerated MAP prepared produce. Campylobacter are zoonotic pathogens, being primarily associated with the intestinal tracts of wild and domestic animals (Thomas et al., 1995) and are distributed throughout the environment through vehicles including birds, surface water and flies. Inappropriate food preparation and handling procedures may lead to the cross-contamination of fresh produce with Campylobacter from uncooked meats, and such errors could have resulted in the identification of MAP prepared vegetable products as sources of infection (Bean and Griffin, 1990; Altrkruse et al., 1994). A Canadian study of 296 fresh-cut MAP vegetable products detected no Campylobacter contamination (Odomeru et al., 1997). 12.2.8 Shigella species The genus Shigella is composed of four species, S. dysenteriae, S. sonnei, S. boydii and S. flexneri, all of which are pathogenic to humans at a low dose of infection. Fruits and vegetables may become contaminated with Shigella via infected food handlers or through the use of contaminated manure and irrigation water (FDA, 2001; Saddik et al., 1985). Several outbreaks of shigellosis have been attributed to contaminated produce (Freudland et al., 1987; see Table 12.1) and a 1986 outbreak of shigellosis was traced back to commercially distributed shredded packaged lettuce (Davis et al., 1988). Despite their mesophilic status, Shigella can survive on lettuce stored at 5oC for seven days (Davis et al., 1988) and on coleslaw at 4oC for 16 days with numbers decreasing slightly during storage (Rafii and Lundsford, 1997). 12.2.9 Viral and protozoan pathogens The significance of viruses with respect to foodborne disease is clear with the inclusion of Norwalk virus, Hepatitis A virus and ‘other viruses’ within the top ten causes of foodborne disease outbreaks in the USA (1983–1987; Cliver, 1997). Outbreaks caused by hepatitis A virus, calicivirus and Norwalk-like viruses have been associated with the consumption of frozen raspberries and strawberries, melons, lettuce, watercress and diced tomatoes (Beuchat, 1996; Hedberg and Osterholm, 1993; Hutin et al., 1999; Lund and Snowdon, 2000; Rosenblum et al., 1990). Viruses can be transmitted by infected food handlers, through the fecal-oral route, and have been isolated from sewage and untreated water used for crop irrigation. Despite their significance, data regarding the effects of food preparation and storage conditions on the survival and infectivity of viruses is extremely limited, partly through the complexity of viral detection assays. Nonetheless, the potential of several viruses to survive on vegetables for periods exceeding their normal shelf-life has been identified (Badawy et al., Reducing pathogen risks in MAP-prepared produce 239 Table 12.2 Foodborne infections linked to the consumption of raw fruits and vegetables Pathogen Product suspected No. of cases Location Reference Bacteria L. monocytogenes Shredded cabbage in coleslaw 41 Canada Schlech et al., 1983 Raw tomatoes, lettuce and celery 20 Boston, US Ho et al., 1986 Cl. botulinum Shredded cabbage in coleslaw 4 Florida, US Solomon et al., 1990 Chopped garlic in oil 37 British Columbia Solomon and Kautter, 1988 Salmonella spp. Sliced watermelon 39 Michigan, US Blostein, 1993 Cantaloupe melon 22 Canada Deeks et al., 1998 Cress sprouts 31 UK Feng, 1997 Mung sprouts 143 UK O’Mahony et al., 1990 Tomatoes 85 Multi-state US Susman, 1999 Tomatoes 174 Multi-state US Tauxe, 1997 E. coli O157:H7 Cantaloupe melon 9 Oregon, US Del Rosario and Beuchat, 1995 Radish sprouts 6561 Japan WHO, 1996 Alfalfa sprouts 108 US CDC, 1997a Lettuce 70 Montana, US Ackers et al., 1998 Lettuce 23 Canada Preston et al., 1997 Shigella sonnei Watermelon 15 Sweden Freudlund et al., 1987 Shredded lettuce 347 Texas Davis et al., 1988 Lettuce 140 Texas Martin et al., 1986 Lettuce 118 Norway, UK, Sweden, Kapperud et al., 1995 Spain Parsley 310 Multi-state US CDC, 1999 Bacillus cereus Soy, mustard and cress sprouts 4 Texas Portnoy et al., 1976 Yersinia enterocolitica Beansprouts 16 US Cover and Aber, 1989 Camylobacter jejuni Salad 330 Canada Allen, 1985 Lettuce 14 Oklahoma, US CDC, 1998a Viruses Hepatitis A virus Raspberries (frozen) 24 Scotland Reid and Robinson, 1987 Strawberries (frozen) 242 + 14 suspect Multistate, US Hutin et al., 1999 Lettuce 103 Florida, US Lowry et al., 1989 Watercress 129 Tennessee CDC, 1971 Diced tomatoes 92 Arkansas, US Lund and Snowdon, 2000 Norwalk virus Melon 206 UK Lund and Snowdon, 2000 Fresh-cut fruit >217 Hawaii Herwaldt et al., 1994 Raspberries (frozen) >500 Finland Lund and Snowdon, 2000 Parasites Cyclospora cayetanensis Raspberries 1465 20 US states & Canada Herwaldt and Ackers, 1997 Raspberries 1012 Multi-state US & Herwaldt and Beach, 1999 Canada Blackberries 104 Canada Herwaldt, 2000 Baby lettuce leaves >91 Florida, US Herwaldt and Beach, 1999 Basil >308 Multi-state US CDC, 1997b Cryptosporidium parvum Green onions 54 Washington CDC, 1998b Giardia Lettuce and onions 21 New Mexico CDC, 1989 1985; Konowalchuk and Speirs, 1975; Sattar et al., 1994). Survival appears to be dependent upon temperature and moisture content (Bidawid et al., 2001; Konowalchuk and Speirs, 1975); however, little information is available on the effects of MAP on virus survival. The protozoan parasites Giardia lamblia, Cyclospora cayetanensis and Cryptosporidium parvum have been the cause of serious foodborne outbreaks involving berries (Herwaldt, 2000; Herwaldt and Ackers, 1997), lettuce and onions (CDC, 1989) and raw sliced vegetables (Mintz et al., 1993). These organisms normally gain access to produce before harvest, usually as a result of contaminated manure or irrigation water and poor hygiene practices by food handlers (Beuchat, 1996). The lack of sensitive methods for determining the survival or inactivation of oocysts has hampered incidence studies and studies focused on the effects of minimal processing and packaging. However, the increase in produce-linked outbreaks due to these organisms (see Table 12.2) indicates that research is needed to examine the behaviour of foodborne protozoan parasites on MAP produce. 12.3 Factors affecting pathogen survival Pathogen survival on produce is influenced by a number of interdependent factors, principally storage temperature, product type/product combinations (e.g. vegetables combined with cooked ingredients), minimal processing operations (e.g. slicing, washing/disinfection), package atmosphere and competition from the natural microflora present on produce. 12.3.1 Storage temperature Storage temperature is the single most important factor affecting survival/growth of pathogens on MAP produce. Storage of produce at adequate refrigeration temperatures, will limit pathogen growth to those that are psychrotrophic; L. monocytogenes, Y. enterocolitica, non-proteolytic Cl. botulinum and A. hydrophila being amongst the most notable. Although psychrotrophic organisms, such as L. monocytogenes, are capable of growth at low temperatures, reducing the storage temperature ( 4oC) will significantly reduce the rate of growth (Beuchat and Brackett, 1990a; Carlin et al., 1995). L. monocytogenes populations remained constant or decreased on packaged vegetables stored at 4oC, while at 8oC, growth of L. monocytogenes was supported on all vegetables, with the exception of coleslaw mix (Francis and O’Beirne, 2001a). Thus even mild temperature abuse during storage permits more rapid growth of psychrotrophic pathogens (Berrang et al., 1989a; Carlin and Peck, 1996; Conway et al., 2000; Farber et al., 1998; Garc?′a- Gimeno et al., 1996; Rodriguez et al., 2000). Mesophilic pathogens, such as Salmonella and E. coli O157:H7, are unable to grow where temperature control is adequate (i.e. 4oC). However, if temperature abuse occurs, they may then grow. Survival of Salmonella in 242 Novel food packaging techniques produce stored for extended periods in chilled conditions may be of concern (Piagentini et al., 1997; Zhuang et al., 1995); Salmonella survived on a range of vegetables for more than 28 days at 2–4oC (ICMSF, 1996). E. coli O157:H7 populations survived on produce stored at 4oC and proliferated rapidly when stored at 15oC (Richert et al., 2000). Reducing the storage temperature from 8 to 4oC significantly reduced growth of E. coli O157:H7 on MAP vegetables; however, viable populations remained at the end of the storage period at 4oC (Francis and O’Beirne, 2001a). The survival of viruses on produce also depends upon temperature. Survival of Hepatitis A virus on lettuce was significantly lower at room temperature than at 4oC (Bidawid et al., 2001). These results are consistent with those of Bagdasaryan (1964), as well as with those of Badawy et al. (1985), who found the greatest survival rates of viruses were at refrigeration temperatures. The behaviour of protozoan parasites on refrigerated produce is not known. However, the increase in incidence of produce-linked outbreaks due to these organisms indicates that research in this area is necessary. Besides its direct effect on pathogen survival/growth, temperature may indirectly affect pathogen growth. Temperature determines the respiration rate of produce, and therefore changes in gas atmospheres within packages, which may influence pathogen growth. Reducing the storage temperature also reduces the growth of the mesophilic spoilage microflora. In the absence of spoilage microflora, high populations of pathogens may be achieved and the item consumed because it is not perceived as spoiled. The elimination or significant inhibition of spoilage microorganisms should not be practised, as their interactions with pathogens may play an integral role in product safety. Guidelines for handling chilled foods, published by the UK Institute of Food Science and Technology (IFST, 1990), recommend a storage temperature range of 0–5oC for prepared salad vegetables, noting that some vegetables may suffer damage if kept at the lower end of this temperature range. Strict control of refrigeration temperature throughout the chill-chain is crucial for maintaining microbiological safety. 12.3.2 Product type/product combinations Produce may include whole or sliced/diced fruits, leaves, stems, roots, tubers or flowers (Burnett and Beuchat, 2001). While all produce items have factors in common, each product has a unique combination of compositional and physical characteristics and will have specific growing, harvesting and processing practices, and storage conditions. Survival/growth of pathogens on produce varies significantly with the type of product (Austin et al., 1998; Carlin and Nguyen-the, 1994; Jacxsens et al., 1999). Dry coleslaw mix was largely unsuitable for L. monocytogenes and E. coli O157:H7 growth while significant growth of the pathogens occurred on shredded lettuce (Francis and O’Beirne, 2001a, b). Product factors that may affect pathogen survival and/or growth include: pH, presence of competitive Reducing pathogen risks in MAP-prepared produce 243 microflora and/or naturally occurring antimicrobials and respiration rate/ packaging interactions. Product pH strongly influences the survival/growth of pathogens. Most vegetables have a pH of 5.0, and consequently support the growth of most foodborne bacteria. Many fruits have acidic pH; however, a number of melons/ soft fruits have pH values 5.0 which will support growth of pathogens (Beuchat, 1996; NACMCF, 1999; Escartin et al., 1989; Lund 1992; Nguyen-the and Carlin, 1994). L. monocytogenes survived and grew on apple slices and cantaloupe melon (Conway et al., 2000; Ukuku and Fett, 2002), and whole tomatoes (Beuchat and Brackett, 1991). Acid tolerance is common in E. coli O157:H7 and Salmonella serotypes and these organisms can survive/grow in acidic produce (Dingman, 2000; Liao and Sapers, 2000; Ukuku and Sapers, 2001; Wei et al., 1995; Zhuang et al., 1995). Some plant tissues have naturally occurring antimicrobials that provide varying levels of protection against pathogens (Lund, 1992; Sofos et al., 1998). The inhibitory effects of raw carrots and carrot juice on growth of L. monocytogenes have been reported (Beuchat et al., 1994; Beuchat and Brackett, 1990b; Jacxsens et al., 1999; Nguyen-the and Lund, 1991). Garlic and onion extracts exhibited antimicrobial properties, red chicory was antagonistic to certain Pseudomonas spp. as well as to A. hydrophila, and cooked cabbage and Brussels sprouts were inhibitory towards Listeria (Beuchat et al. 1986; Beuchat and Brackett, 1990b; Jacxsens et al., 1999; Nguyen-the and Carlin, 1994). MAP produce harbours a large and diverse microflora. Effects of competition between the indigenous microflora and pathogens on MAP produce may play an important role in product safety (see Section 12.3.5). Beansprouts did not support good growth of L. monocytogenes or E. coli O157:H7, due presumably to competition from high populations of background microflora, inhibition from the relatively high in-pack CO 2 levels (25–30%) and the more limited nutrient availability of intact vegetables (Francis and O’Beirne, 2001a). Minimally processed produce may be combined with cooked ingredients. Growth of L. monocytogenes on raw endive was probably limited by nutrient availability, but reached higher numbers when sweetcorn was added (Carlin et al., 1996b; Nguyen-the et al., 1996). The addition of cooked products to raw vegetables supplied a source of nutrients and permitted rapid growth of both spoilage and pathogenic populations on such products (Thomas and O’Beirne, 2000). 12.3.3 Minimal processing operations The unit operations employed during the production of minimally processed produce (handling, peeling, slicing, washing, packaging) cause the destruction of surface cells, affect product respiration rate and pH, and release nutrients and possibly antimicrobial substances from the plant cells (Brackett, 1994), which will in turn affect the behaviour of pathogens. 244 Novel food packaging techniques In general, pathogens will not grow on uninjured surfaces of fresh intact produce; however, cutting or slicing facilitates contamination by pathogens and subsequent survival and/or growth. Injuries to the wax layer, cuticle and underlying tissues increased bacterial adhesion and growth (Han et al., 2000a, 2001; Seo and Frank, 1999; Takeuchi and Frank, 2001; Takeuchi et al., 2000). Consequently, minimising damage throughout harvesting and processing reduces the chances of pathogen contamination, penetration and growth (Liao and Cooke, 2001). Pathogens can become attached to processing equipment (slicers, shredders) and once attached (biofilms) are very difficult to remove by chemical sanitisers (Bremer et al., 2001; Frank and Koffi, 1990; Garg et al., 1990; Jo¨ckel and Otto, 1990; Nguyen-the and Carlin, 1994). Indeed, L. monocytogenes has been recovered from the environment of processing operations used to prepare minimally processed vegetables (Zhang and Farber, 1996), highlighting the importance of strict hygiene during processing. Recommendations implemented to ensure quality and safety of produce relate to good manufacturing practices (see Section 12.4; Koek et al. (1983), microbial specifications for the processed product, and proper storage conditions (Nguyen-the and Carlin, 1994). Washing/antimicrobial dipping Washing in tap water removes soil and other debris, some of the surface microflora, and cell contents and nutrients released during slicing that help support growth of microorganisms (Bolin et al., 1977). However, water washing had minimal effects on microorganisms on fresh produce (Beuchat, 1992; Nguyen-the and Carlin, 1994; Brackett, 1987; Adams et al., 1989; Izumi, 1999) and due to the re-use of wash water in industry may result in cross- contamination of food products and food-preparation surfaces (Beuchat and Ryu, 1997; Brackett, 1992; Beuchat, 1996; Garg et al., 1990). A variety of antimicrobial wash solutions have been used to reduce populations of microorganisms on fresh produce. The effectiveness of disinfection depends on a number of factors including: (i) type of treatment, (ii) type, numbers and physiology of the target microorganism(s), (iii) product type, (iv) disinfectant concentration, (v) pH of the disinfectant solution, (vi) exposure time, (vii) temperature of washing water and (viii) general sanitation of plant and equipment (Adams et al., 1989; Best et al., 1990; El-Kest and Marth, 1988a,b). Chlorine (50–300ppm) is the most frequently used disinfectant for fresh fruits and vegetables; added to water as a solid, liquid or gas (Adams et al., 1989; Anon., 1973; Beuchat and Ryu, 1997; Lund, 1983). Total microbial populations were reduced about 1000-fold when lettuce was dipped in water containing 300ppm total chlorine, but no effect was seen against microbial populations on red cabbage or carrots (Garg et al., 1990). Generally, no more than 2- to 3-log 10 reductions of bacteria on produce after chlorine treatment have been reported (Adams et al., 1989; Beuchat, 1992; 1999). The effects of chlorine in removing pathogens from produce have been studied. L. monocytogenes counts on Brussels sprouts were reduced approxi- Reducing pathogen risks in MAP-prepared produce 245 mately 100-fold by chlorine treatment (200mg/l), 10-fold more than those treated with water (Brackett, 1987). The maximum log 10 reductions of L. monocytogenes, after treatment with chlorine (200ppm), were 1.7 for lettuce and 1.2 for cabbage (Zhang and Farber, 1996). Dipping coleslaw and lettuce in a chlorine solution (100ppm) reduced initial L. innocua and E. coli populations, but resulted in enhanced survival during extended storage at 8oC (Francis and O’Beirne, 2002). Chlorine (100–200ppm) was only marginally effective at reducing E. coli levels on lettuce tissue surfaces (Beuchat, 1999), apple surfaces (Wisniewsky et al., 2000; Wright et al., 2000) and broccoli florets (Behrsing et al., 2000). Salmonella populations on alfalfa sprouts were reduced by about 2 log 10 CFU/g after treatment with 500ppm chlorine, and to undetectable levels after treatment with 2,000ppm chlorine (Beuchat and Ryu, 1997). Ten-minute exposures of Y. enterocolitica on shredded lettuce to 100 and 300ppm chlorine resulted in population reductions of 2–3 log 10 cycles (Escudero et al., 1999). In the same study, a combination of 100ppm chlorine and 0.5% lactic acid inactivated Y. enterocolitica by >6 log cycles, suggesting that Y. enterocolitica may be more sensitive to chlorine than other pathogens. Chlorine, used at concentrations currently permitted in the industry to wash fresh produce, cannot be relied upon to eliminate pathogens (see Chapter 23). The ineffectiveness of chlorine treatment may be due to a number of factors. The hydrophobic nature of the waxy cuticle on produce protects surface contaminants from exposure to chlorine which does not penetrate or dissolve these waxes/oils (Adams et al., 1989). In addition, microbial cells may become embedded in crevices, creases or injured tissues and are inaccessible to chlorine treatments (Adams et al., 1989; Lund, 1983; Koseki et al., 2001; Seo and Frank, 1999; Takeuchi and Frank, 2000, 2001). Organic matter (fruit and vegetable components) neutralises chlorine, rendering it inactive against microorganisms (Beuchat, 1996; Beuchat et al., 1998; Lund, 1983). It is important to sanitise injured surfaces before cutting as once cut or injured surfaces are contaminated by pathogens, it is very difficult to remove these attached and growing microorganisms. The most useful effect of chlorine may be in inactivating vegetative cells in washing water and on equipment during processing as part of a HACCP system, thus avoiding build-up of bacteria and cross-contamination (Wilcox et al., 1994). A concern regarding the use of chlorine dips is that pathogens may not be fully eliminated by commercial treatments, while at the same time natural competitive organisms may be removed. L. monocytogenes inoculated onto disinfected (10% hydrogen peroxide) endive leaves grew better than on water- rinsed produce (Carlin et al., 1996b) and dipping lettuce in a chlorine (100ppm) solution followed by storage at 8oC, significantly enhanced Listeria growth compared with undipped samples (Francis and O’Beirne, 1997). Disinfection before contamination with the pathogen occurs may increase growth of the pathogen because populations of competing microflora have been removed (Bennik et al., 1996). Therefore, temperature management (i.e. 4oC) after reduction of microbial populations is crucial for microbial safety. 246 Novel food packaging techniques Due to the ineffectiveness of chlorine in removing pathogens from produce and increasing concern over the production of chlorinated organic compounds and their impact on human and environmental safety, a variety of other disinfectants, including acidic electrolysed water (Park et al., 2001), peroxyacetic acid (Park and Beuchat, 1999), chlorine dioxide (Zhang and Farber, 1996), hydrogen peroxide (Sapers and Simmons, 1998), organic acids (Karapinar and Gonul, 1992), trisodium phosphate (Zhang and Farber, 1996) and ozone (Burrows et al., 1999) have been evaluated (Beuchat, 1999) (see Chapter 23). However, none of the sanitiser treatments tested is likely to be totally effective against all pathogens, and behaviour of pathogens during subsequent storage remains unpredictable (Beuchat and Ryu, 1997; Escudero et al., 1999; Park and Beuchat, 1999; Zhang and Farber, 1996). Viruses and protozoan cysts on fruits and vegetables generally exhibit higher resistance to disinfectants than do bacteria or fungi (Beuchat, 1998). Feline caliciviruses were very resistant to commercial disinfectants; however, peroxyacetic acid and H 2 O 2 were effective at decontaminating strawberries and lettuce when used at four-fold higher concentrations than generally recommended (Gulati et al., 2001). Treatment of Cryptosporidium parvum oocysts with 1ppm ozone for five minutes resulted in <1 log 10 inactivation (Korich et al., 1990). 12.3.4 Package atmosphere When a sliced product is packaged, it continues respiring thereby modifying the gas atmosphere inside the package. Ideally, O 2 levels will fall from the 21% found in air to 2–5%, and CO 2 levels will increase to the 3–10% range. Atmospheres within MA packages might be cause for public health concern in at least three ways. The atmospheres and refrigeration temperatures employed may inhibit the development of some spoilage aerobic microorganisms (Daniels et al., 1985; Farber, 1991). Consequently, their suppression may facilitate pathogen survival/ growth, without the product showing obvious signs of spoilage. Secondly, MAP increases the shelf-life of products, thus increasing the time available for pathogens to grow. Over-extending the shelf-life may allow development of significant populations, particularly if combined with exposure to even modest abuse temperatures. Thirdly, although the low levels of O 2 (2–5%) within packages (e.g. at 4oC) should inhibit growth of obligate anaerobes such as Cl. botulinum, if packages are subjected to temperature abuse, they may become anaerobic as a result of increased product respiration. This could enable growth and toxin production by Cl. botulinum to occur (see Section 12.2.2). In addition to the target atmospheres described above, there is evidence of significant incidence of ‘unintended’ atmospheres in commercial practice. Where the gas permeability of packaging films is insufficient, produce with high respiration rates may generate MAs which are anoxic and/or contain high levels (>20%) of CO 2 . Of particular concern with refrigerated MAP produce is the growth of psychrotrophic, facultatively anaerobic and microaerophilic microorganisms, Reducing pathogen risks in MAP-prepared produce 247 which can tolerate refrigeration temperatures and low O 2 atmospheres (Bennik et al., 1995), and a number of studies have indicated that MAP may select for such pathogens (Hintlian and Hotchkiss, 1986; Brackett, 1994; Beuchat and Brackett, 1990a; Kallender et al., 1991). There are inconsistencies in the literature regarding the effects of MAP on the growth of L. monocytogenes. Numerous researchers have shown that survival of L. monocytogenes on produce remains largely unaffected by MAP (Amatanidou et al., 1999; Berrang et al., 1989b; Beuchat and Brackett; 1990a, 1991; Conway et al., 1998, Jacxsens et al., 1999). However, in other work, nitrogen flushing combined with storage at 8oC enhanced the growth of L. monocytogenes on shredded lettuce (Francis and O’Beirne, 1997) and shredded chicory salads (Ringle′ et al., 1991). Several studies have demonstrated that A. hydrophila can grow rapidly on vegetables stored at 4–5oC and MAP does not significantly affect its growth (Berrang et al., 1989a). Austin et al. (1998) found that some samples of MAP vegetables appeared organoleptically acceptable when Cl. botulinum toxin was detected (see Section 12.2.2). Salmonella and E. coli O157:H7 can grow under MAP conditions; however, there is insufficient information available on whether atmospheres inhibit or enhance their growth. CO 2 had little or no inhibitory effect on growth of E. coli O157:H7 on shredded lettuce stored at 13 or 22oC, and growth potential was increased in an atmosphere of O 2 /CO 2 /N 2 : 5/30/65, compared with growth in air (Abdul-Raouf et al., 1993; Diaz and Hotchkiss, 1996). A recent study, investigating C. jejuni survival on MAP vegetables found that refrigeration temperatures in combination with a MA (2% O 2 , 18% CO 2 and 80% N 2 ) were favourable for the organism (Tran et al., 2000). The highest rates of Hepatitis A virus survival on lettuce stored at 4oC (12 days) was observed under 70% CO 2 /30% N 2 and 100% CO 2 (Bidawid et al., 2001). In ‘unintended’ atmospheres, high CO 2 levels may develop within packages. Carlin et al. (1996a) examined the survival of L. monocytogenes on chicory leaves stored at 10oC in air, or under 10%, 30% or 50% CO 2 , with 10% O 2 and found that L. monocytogenes grew better as the concentration of CO 2 increased. The growth rate of A. hydrophila decreased with increasing CO 2 concentrations, but maximum population densities were not affected by CO 2 concentrations of up to 50% (Bennik et al., 1995). Novel, alternative techniques to low O 2 MAP are the use of high O 2 (i.e. >70% O 2 ) atmospheres and noble gases (Day, 1996; see Chapter 10). In an agar-based study to investigate the effects of high O 2 (90%) and moderate CO 2 (10–20%) concentrations on foodborne pathogens at 8oC, Amanatidou et al. (1999) noted inhibitory action against L. monocytogenes, A. hydrophila, S. Typhimurium, S. Enteritidis and E. coli. Studies to determine the behaviour of Y. enterocolitica on MAP produce have not been published and information describing the survival of Campylobacter spp. on MAP produce is extremely limited. In addition, the behaviour of protozoan parasites under MAP is not known. Therefore, more research to determine the survival of these and other pathogens on MAP produce is warranted. 248 Novel food packaging techniques 12.3.5 Competition between the indigenous microflora and pathogen MAP produce harbours large populations of native microorganisms including pseudomonads, lactic acid bacteria (LAB) and Enterobacteriaceae (Francis et al., 1999; Nguyen-the and Carlin, 1994). The background microflora provide indicators of temperature abuse largely by causing detectable spoilage, and can vary significantly for each product and during storage. LAB can exert antibacterial effects due to one or more of the following mechanisms: lowering the pH (Raccach and Baker, 1979); generating H 2 O 2 (Price and Lee, 1970); competing for nutrients (Iandolo et al., 1965); and possibly by producing antimicrobial compounds, such as bacteriocins (Arihara et al., 1993; Harris et al., 1989; Klaenhammer, 1988). Cai et al. (1997) reported that a large portion of LAB isolates from bean sprouts inhibited the growth of L. monocytogenes. Strains of LAB were reported to inhibit A. hydrophila, L. monocytogenes, S. Typhimurium, and Staphylococcus aureus on vegetable salads (Vescovo et al., 1996). Competition from LAB may limit pathogen growth on produce, but there is insufficient data available to prove this conclusively. Various researchers have reported antagonism by the native microflora of vegetables against Listeria (Francis and O’Beirne, 1998a,b; Liao and Sapers, 1999). Reducing the background microflora of endive leaves (Carlin et al., 1996b) and shredded lettuce (Francis and O’Beirne, 1997) resulted in enhanced growth of Listeria. A mixed bacterial population isolated from endive or lettuce reduced Listeria growth in vegetable media (Carlin et al., 1996b; Francis and O’Beirne, 1998a, b). However, the inhibitory effects were dependent on gas atmosphere; in 3% O 2 (balance N 2 ) growth of the mixed population was inhibited while L. monocytogenes proliferated (Francis and O’Beirne, 1998a). Fluorescent pseudomonads have previously been shown to stimulate growth of L. monocytogenes in various foods, due to the release of potential nutrients by pseudomonads (Nguyen-the and Carlin, 1994; Liao and Sapers, 1999; Marshall and Schmidt, 1991). Bennik et al. (1996) found that strains of fluorescent pseudomonads slightly reduced final population densities of L. monocytogenes in an endive leaf medium. P. fluorescens and P. viridiflava inhibited growth of L. monocytogenes on potato slices while Erwinia carotovora and Xanthomonas campestris did not affect its growth (Liao and Sapers, 1999). Enterobacter isolates (Enterobacter cloacae, Enterobacter agglomerans) significantly reduced L. monocytogenes growth during storage on a model medium; however, the inhibitory activities of Enterobacter spp. decreased as the concentration of CO 2 increased (Francis and O’Beirne, 1998a). Del Campo et al. (2001) also found that Enterobacteriaceae (Enterobacter agglomerans, Rhanella aquatilis) reduced maximum population densities of L. monocytogenes in minimal media, presumably due to competition for glucose and/or amino acids. Ukuku and Fett (2002) reported that the native microflora of cantaloupe melon, especially the yeast and mould populations, might have out-competed L. monocytogenes for colonisable space and available nutrients, thus resulting in the decline of populations of L. monocytogenes. Reducing pathogen risks in MAP-prepared produce 249 Growth of the background microflora also significantly affected the growth and toxigenesis of Cl. botulinum in refrigerated foods (Hutton et al., 1991; Hauschild, 1989) and Larson and Johnson (1999) demonstrated the ability of spoilage microflora to protect against Cl. botulinum outgrowth. A. hydrophila has been reported to be a poor competitor with LAB and other spoilage organisms (Palumbo and Buchanan, 1988). MAP and chill temperatures, combined with the use of a Lactobacillus casei inoculum, reduced growth of A. hydrophila on vegetables such as lettuce (Vescovo et al., 1997). Competitive microflora had a significant effect on the growth of E. coli O157:H7 in broth media; Hafnia alvei significantly inhibited the growth of E. coli O157:H7 at 37oC, whereas Pseudomonas fragi inhibited growth of the pathogen at 15oC (Duffy et al., 1999). Little is known about the mechanism by which Salmonella manages to compete with natural microflora and survive on plant products (Liao and Cooke, 2001). Wells and Butterfield (1997) demonstrated that salmonellae grew better on vegetables when co-cultured with Erwinia carotovora or P. viridiflava, two major causes of bacterial soft-rot. Complex interactions with the indigenous microflora may have significant effects on survival/ growth of pathogens. More research needs to be done to examine the influence of gas atmospheres, background microflora and storage temperatures on the survival/growth of pathogens, including foodborne viruses and protozoan parasites on MAP produce in order to ensure that novel mild preservation technology can continue to be applied safely. 12.3.6 Minimal processing and stress responses Pathogenic bacteria can respond or adapt to sub-lethal stresses encountered in minimal processing in ways that increase their resistance to more severe treatments and enable better survival in foods (Buncic and Avery, 1998; Abee and Wouters, 1999; Gahan and Hill, 1999). Apart from the enhanced survival in foods and increased resistance to subsequent food processing/preservation treatments, adapted or hardened pathogens may also have enhanced virulence (Abee and Wouters, 1999; Gahan and Hill, 1999; Rouquette et al., 1998). Two of the best studied adaptive tolerance responses are to heat (heat stress response) and to acid (acid tolerance response, ATR). Acid adapted L. monocytogenes, Salmonella and E. coli O157:H7 survived significantly better in acidic foods such as salad dressing and fruit juices, when compared to non- adapted cells (Gahan et al., 1996; Leyer and Johnson, 1992). Acid adaptation induces acid tolerance to more severe or normally lethal acid, but it can also induce cross-protection against other environmental stresses such as thermal and osmotic stress (Leyer and Johnson, 1993; Lou and Yousef, 1997; O’Driscoll et al., 1996). Equally other stresses can induce acid tolerance. Acid adaptation enhanced survival of L. monocytogenes during storage in packages of vegetables which had relatively high in-pack CO 2 levels (25–30% in MAP coleslaw and bean sprouts; Francis and O’Beirne, 2001b). E. coli O157:H7 survived in an acidic environment better at 4oC than at 10oC, which implies that induction of 250 Novel food packaging techniques acid tolerance may enhance resistance to low temperature (Conner and Kotrola, 1995). 12.3.7 Implications of strain variation among pathogens The selection of strain(s) of a particular pathogen to be used in survival studies is extremely important as different strains may behave differently on MAP produce. Unpublished work carried out by the authors has shown that strains of L. monocytogenes differ significantly in their inherent ability to survive/grow on MAP vegetables. In addition, there was significant variation among strains in their inherent stress resistance characteristics; some strains may be more resistant to the stressful conditions encountered in foods and during food processing. Although the response of L. monocytogenes to food related growth factors (e.g. temperature, pH, gas atmosphere) has been studied extensively, in most studies only one strain has been tested. In studies where multiple isolates were examined, significant strain variation in resistance existed among L. monocytogenes isolates (Begot et al., 1997; Barbosa et al., 1994; Buncic et al., 2001; Dykes and Moorhead, 2000; Mackey et al., 1990; Palumbo et al., 1995) and there were some differences between serotypes (Davies and Adams, 1994; Embarek and Huss, 1993; So¨rqvist, 1994). Junttila et al. (1988) reported that there were differences in ability of L. monocytogenes strains to grow at low temperatures, with strains in the serotype 1/2 capable of growth at colder temperatures than strains of serotype 4b. Evidence also suggests that L. monocytogenes strains differ at the molecular level; however, little is known of the attributes that contribute to the ability of certain strains to cause disease, an ability that can vary significantly between individual strains (Barbour et al., 1996; Del Corral et al., 1990; Tabouret et al., 1991; Brosch et al., 1993; Farber and Peterkin, 1991; Rocourt, 1994). The diversity of the genus Salmonella has been observed in many different forms, from genetic to physiological observations. The ability of E. coli O157:H7 to tolerate heat was strain dependent (Clavero et al., 1998; Duffy et al., 1999) and survival of E. coli O157:H7 on vegetables depended on bacterial strain and product type (Francis and O’Beirne, 2001a). Different strains of pathogens may respond differently to treatments including mild acid, low temperature and gas atmosphere, which may result in variations in the ability of surviving populations to cause human disease (Buncic et al., 2001). 12.4 Improving MAP to reduce pathogen risks The pathogen risks from MAP produce cannot be totally eliminated, but they can be minimised by applying best practice at every stage – agricultural production, pre-processing, processing, distribution and final use. At all stages, strategies to minimise contamination by pathogens, product storage at 4oC and Reducing pathogen risks in MAP-prepared produce 251 education/training of workers and consumers are important recurring themes. Clearly, Good Agricultural Practices (GAP) and Good Manufacturing Practices (GMP) need to be put in place to minimise hazards, and many Codes of Practice have been published by national agencies (e.g. FSAI, 2001) and industry sectors. However, there may be insufficient data available on which to base a comprehensive validated Hazard Analysis and Critical Control Points (HACCP) Programme for most produce items (NACMCF, 1999). 12.4.1 Production of raw materials Agricultural production practices can have major implications for contamination of raw produce with pathogens (Gorny and Zagory, 2000), and producer awareness of their role in assuring food safety is vitally important. This is an extremely complex arena with great diversity in crop production methods, scale, environmental factors, etc. (FDA, 1998). Land subject to flooding or on which animals have grazed should be avoided (Brackett, 1999). Improperly composted sewage or animal manure should not be applied to land where vegetables for processing are grown. However, persistence of L monocytogenes has been demonstrated even in treated sewage sludge (Al Ghazali and Al Azawi, 1986). Proximity to animal production facilities may also be a significant cross-contamination hazard. Irrigation should be carried out with clean water, pretreated if necessary (Robinson and Adams, 1978) and applied as trickle irrigation at ground level rather than as an overall spray (NACMCF, 1999). However, serious deficits in water quality and availability exist globally, with water pollution from sewage and animal production facilities posing serious problems. Workers involved in harvesting and handling should be trained in the principles of good sanitation and provided with adequate washing/toilet facilities in fields and packhouses (Brackett, 1999). Harvesting equipment should be thoroughly cleaned and sanitised. Birds such as gulls and pigeons, wild animals, domestic animals and insects should be excluded from packhouses and processing areas (NACMCF, 1999). Wild birds are known to disseminate Campylobacter, Salmonella, Vibrio cholera, Listeria species and E.coli O157, apparently picked up from feeding on garbage, sewage, etc; control of preharvest contamination of produce by wild birds is particularly difficult (Beuchat and Ryu, 1997). Increasing globalisation of produce supplies poses serious new challenges (Tauxe, 1997) and knowledge of contamination levels in imported produce is minimal (Beuchat and Ryu, 1997). The only rational solution is the extension of the requirement for GAPs to wherever primary production takes place. 12.4.2 Minimal processing Based on the data discussed in Section 12.3, processing can be geared to minimise opportunities for pathogen contamination and growth. Starting at harvest, bruising and cutting should be minimised prior to processing (Liao and 252 Novel food packaging techniques Cooke, 2001). Immediately prior to processing, preliminary decontamination should be carried out by removing outer leaves, soil, etc., from produce using sharp sanitised knives for any cutting. Peeling, cutting, shredding, etc., should be carried out with equipment designed to cause the minimum of tissue disruption, as severe processing may facilitate more effective contamination and subsequent growth by pathogens (Gleeson et al., 2002). Severe processing may also reduce the effectiveness of subsequent anti-microbial treatments (Han et al., 2000a; Han et al., 2000b; Liao and Cooke, 2001; Liao and Sapers, 2000; Takeuchi and Frank, 2000, 2001). GMP should include effective surface and machine sanitising to eliminate the risk of pathogen contamination from the processing environment or from machines used in processing (Zhang and Farber, 1996; Nguyen-the and Carlin, 1994). Food safety experts should be consulted by engineers designing processing equipment to ensure ease of sanitisation (Beuchat and Ryu, 1997). Human contact should be eliminated or minimised to reliable trained staff. Although its benefits are questioned (Brackett, 1999), anti-microbial dipping is probably a valuable tool for reducing numbers of potential pathogens (Beuchat and Ryu, 1997). State-of-the-art effective systems are available and should be used. Some of these greatly reduce the levels of chlorine needed (Varoquaux, 2001). Special care should be exercised to avoid contamination after dipping. Post- processing risks introduced by anti-microbial dips (Francis and O’Beirne, 1997; Carlin et al., 1996b; Bennik et al., 1996) should be addressed in HACCP protocols: the most important of these are measures to ensure that products are stored at 4oC at all times and the use of conservative use-by dates. Where alternatives to chlorine are being introduced, any differences in their anti-microbial effects should be understood and taken into account. 12.4.3 Modified atmosphere packaging Packaging materials must be carefully selected to ensure that their gas permeability properties match the respiration rates of the products being packaged. This is necessary in order to achieve package atmospheres within the technically useful range of 2–5% O 2 and 3–10% CO 2 (Cliffe-Byrnes et al., 2003; Barry-Ryan et al., 2000). Technical advice from researchers and packaging suppliers is essential (see Section 12.6), though user-friendly software may be developed to assist industry in the future. Poor ‘package-product compatibility’ will result in the creation of unintended atmospheres with uncertain microbiological implications (Bennik et al., 1998). The use of coatings with gas barrier properties can be a feature of MAP produce (Guilbert et al., 1996), but more information is needed on their effects on internal atmospheres. Other novel elements of MAP include the use of high oxygen and noble gas enriched atmospheres (see Chapter 10). While atmospheres with 80% oxygen have been used in MAP of fresh meat for a few decades and appear safe, less is known about the effects of noble gases such as argon on microbial ecology. The microbial quality of the final packaged Reducing pathogen risks in MAP-prepared produce 253 product should be monitored to ensure that it complies with international guidelines (see Francis et al., 1999). 12.4.4 Distribution and final use Ensuring that temperatures are kept at or below 4oC throughout the cold chain is essential for microbial safety and requires considerable attention to detail. Refrigerated distribution requires suitably designed vehicles, properly loaded to allow for air movement (Brackett, 1999). At supermarket level, LeBlanc et al. (1996) found 90% of produce items above 4 0 C in supermarket chill cabinets. Problems can also arise at consumer level where products are held for extended periods in cars or experience elevated temperatures in (poorly operating) domestic refrigerators. Time temperature indicators embedded in the packaging may have a significant role in ensuring that safe storage temperatures are used (see Chapter 6). Distributors, retailers and consumers must be educated on the importance of low storage temperatures. Consumers also need to be educated on the nature of minimal processing technologies for fresh foods, in particular that consumption of apparently fresh food beyond its use-by date is potentially hazardous. General principles of good hygiene must apply throughout the distribution chain, particularly avoiding cross-contamination. For example, refrigerated trucks carrying vegetables on an outward journey may be used to ‘backhaul’ animals or raw meats (Brackett, 1999). Truck use needs to be monitored and vehicles appropriately sanitised. In the food service sector training of operatives is important since many of these are teenagers and may require food hygiene training within the school system, as they receive little food preparation experience in the modern home (Beuchat and Ryu, 1997). 12.4.5 HACCP strategies While HACCP principles are being applied by many growers, manufacturers, and distributors based on current knowledge, the US National Advisory Committee on Microbiological Criteria for Foods claim that there is insufficient evidence to put in place a comprehensive validated system for fresh produce (NACMCF, 1999). Model farm-to-table HACCP protocols have been developed for only a few commodities (sprouted seeds, shredded lettuce and tomatoes) but even these have not been completely validated. According to NACMCF, further research is needed to provide data and technology for the validated control measures needed for GAP and GMP. 12.5 Future trends The recent rapid growth in the volume of produce consumption and in the globalisation of sourcing can be expected to continue. There will be improved information on emerging and existing pathogens and their interaction with 254 Novel food packaging techniques production and processing technologies. There will be greater application of new and existing technology and of best practice. 12.5.1 Production of raw materials Greater emphasis can be expected on the development and application of GAP protocols, particularly for use of water and manure, for worker hygiene and for transportation of produce. Serious efforts will be made to apply these protocols to production in developing as well as industrialised countries. These initiatives should result in a safer, more reliable raw material stream. 12.5.2 Processing, packaging, and distribution Current interest in alternatives to chlorine dipping are likely to result in novel chemical treatments and the application of physical treatments such as UV radiation (see Chapter 23). Ionising radiation may also be used either alone or in combination with other treatments, as a means of extending the shelf-life of produce (Diehl, 1995; Langerak, 1978). Doses in the range of <1 to 3 kGy have been shown to reduce or eliminate populations of pathogens and postharvest spoilage organisms on produce (Farkas, 1997) and salmonellae were not recovered from alfalfa sprouts irradiated with 0.5 kGy (Rajkowski and Thayer, 2000). However, despite the efficiency, safety and suitability to products with surface contamination (O’Beirne, 1989) the use of irradiation will depend on its acceptance by consumers. Greater use of edible coatings (e.g. sucrose polyesters of fatty acids, cellulose derivatives, etc.) to food surfaces can be expected (Krochta and De Mulder- Johnston, 1997; Baldwin et al., 1995). Edible coatings (e.g. hydroxypropyl methylcellulose) can extend shelf-life, and with the inclusion of anti-microbials, reduce the potential growth of pathogens (Zhuang et al., 1996). Other additional novel processing steps may be introduced such as inoculation of MAP produce with organisms inhibitory to one or more pathogens. For example, strains of LAB inhibited A. hydrophila, L. monocytogenes, S. typhimurium, and Staphylococcus aureus on vegetable salads (Vescovo et al., 1996) and use of a Lactobacillus casei inoculum, reduced growth of A. hydrophila on MAP vegetables such as lettuce (Vescovo et al., 1997). More reliable package atmospheres can be expected as a result of improved materials, temperature-responsive smart packaging and better software to define gas permeability requirements for individual products. There will be widespread commercial application of active packaging with anti-microbial and other properties. Stress responses of pathogens and cross-protection must be considered when current food processing technologies are being modified or new ones developed. These responses are particularly significant in minimal processing/packaging technology where the imposition of one sub-lethal stress may lead to the induction of multiple stress responses that may reduce the efficacy of later Reducing pathogen risks in MAP-prepared produce 255 treatments (Hill et al., 1995). Strategies to prevent such stress responses would facilitate the development of improved procedures for prevention of pathogen survival and growth. For example, new decontamination technologies will be developed which provoke minimal levels of stress response. More generally, micro-array technology will be used to assess the response of both plant materials and pathogens to processing and storage regimes. In relation to temperature control, greater use of IT can be expected in wireless and internet based data collection/operator alerting systems such as those developed by Freshloc Technologies Inc. for monitoring product temperatures during transportation. 12.5.3 Research Research trends driven by the needs of this sector include greater understanding of emerging pathogens, particularly viruses and protozoan parasites; greater understanding of processes of produce contamination generally, and of how to prevent them; the development of new effective decontamination technologies; development and application of active and intelligent packaging. In order to improve surveillance for food-borne illness, there is a need for greater use of molecular techniques for sub-typing of pathogens (serotyping/molecular typing). This technology can help establish sources/points of contamination, links between geographically isolated outbreaks of food poisoning with a common source (NACMCF, 1999), and provide other types of data which will help develop HACCP protocols which can be validated. 12.6 Sources of further information and advice Campden and Chorleywood Food Research Association (1996), Code of practice for the manufacture of vacuum and modified atmosphere packaged chilled foods with particular regards to the risks of botulism, Guideline No. 11. Campden and Chorleywood Food Research Association (1992), Guidelines for the good manufacture and handling of modified atmosphere packed food products, Technical Manual No. 34. Codex Alimentarius http://www.fao.org/es*/esn/codex/ Food and Drug Administration (1998), Guide to minimize microbial food safety hazards for fresh fruits and vegetables. http:www.foodsafety.gov/~dms/ prodguid.html Food and Drug Administration (2000), Kinetics of microbial inactivation for alternative food processing technologies. http:vm.cfsan.fda.gov/~comm/ ift-toc.html Food Safety Authority of Ireland (FSAI, 2001), Code of practice for food safety in the fresh produce supply chain in Ireland, Code of practice No. 4. ISBN 0953918343. 256 Novel food packaging techniques Institute of Food Science and Technology (1990), Guidelines for the handling of chilled foods. ISBN 0905367073. Institute of Food Science and Technology (1992), Guidelines to good catering practice. ISBN 090536709X. 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