Bacillus cereus Soy, mustard and cress sprouts Texas Portnoy et al., 1976 Cover and Aber. 1989 Campylobacter jejuni Allen, 1985 Lettuce Oklahoma, US CDC. 1998a Hepatitis A virus Raspberries(frozen) Scotland Reid and robinson. 1987 Strawberries(frozen 42+ 14 suspect Multistate, US Hutin et al. 1999 Diced tomatoes Norwalk virus Melot Lund and Snowdon, 2000 Fresh-cut fruit Raspberries(frozen) >500 Finland Lund and Snowdon, 2000 Cyclospora cayetanensis Raspberries 20 US states Canada Herwaldt and Ackers. 1997 Raspberries Herwaldt and beach, 199 Blackberries Herwaldt. 2000 truce leave Herwaldt and Beach, 1999 Basil Multi-state US CDC. 199 Cryptosporidium parvum Green onions CDC. 1998b Lettuce and onions New mexico CDC.1989
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
242 Novel food packaging techniques 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 food borne protozoan parasites on MAP produce 12.3 Factors affecting 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(<4C)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 4C, while at 8.C, 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: Carl lin and Peck, 1996 Conway et al, 2000; Farber et al. 1998: Garcia Gimeno et al, 1996; Rodriguez et al, 2000) Mesophilic pathogens, such as Salmonella and E coli o157: H7, are unable row where temperature control is adequate (i.e. <4C).However, if temperature abuse occurs, they may then grow. Survival of salmonella
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; Garcı´aGimeno 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
Reducing pathogen risks in MAP-prepared produce 243 produce stored for extended periods in chilled conditions may be of concern (Piagentini et al, 1997, Zhuang et al., 1995); Salmonella survived on vegetables for more than 28 days at 2-4C (ICMSF, 1996). E. coli O157: H7 populations survived on produce stored at 4C and proliferated rapidly when stored at 15.C (Richert et al., 2000). Reducing the storage temperature from 8 to 4C 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 4C(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 0-5C 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
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
244 Novel food packaging 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 0157: H7 and Salmonella serotypes and these organisms can survive/grow in acidic produce(Dingman, 2000, Liao and Sapers, 2000, Ukuku and Saper 2001; Wei et al., 1995; Zhuang et al, 1995) Some plant tissues have naturally occurring antimicrobials that provide arying 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 w tagonistic 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 CO2 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
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 CO2 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
Reducing pathogen risks in MAP-prepared produce 245 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 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 Koff, 1990; Garg et al., 1990, Jockel 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 debri microflora, and cell contents and nutrients released support growth of microorganisms(Bolin et al., 1977) had minimal effects on microorganisms on fresh produce(Beuchat, 1992 Nguyen-the and Carlin, 1994; Brackett, 1987, Adams et al, 1989; Izumi, 1999) d 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-l0g10 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 appro
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 crosscontamination 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-log10 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 approxiReducing pathogen risks in MAP-prepared produce 245