D CONTROL (cooked crustaceae, frozen or chilled)

Signiﬁcant hazards a r Staph. aureus (post-heating contamination). r Bacterial enteric pathogens (post-heating contamination).

r Viral enteric pathogens (post-heating contamination). r Cl. botulinum (mesophilic in canned crab meat).

Control measures

Initial level (H 0 )

r Not applicable.

Reduction (Σ R)

r Heating/cooking destroys pathogens.

Increase (Σ I)

r Observe GHP to avoid cross-contamination. r Cold storage of canned crab meat.

Testing

r Staph. aureus in cooked crustaceans. r Salmonellae.

Spoilage

r Time × −temperature.

a Under particular circumstances, other hazards may need to be considered.

Hazards considered. As outlined in the section on fresh crustaceans, aquatic toxins, histamine, and parasites are not major safety issues. Pathogenic bacteria, both indigenous and contaminating, are killed by the cooking step leaving a product, which in principle is sterile. As cooked crustaceans typically are handled both by peeling machines and manually following the cooking step, contamination with human borne pathogens, e.g. Staph. aureus, salmonellae, or virus may occur. Also, cross-contamination by Vibrio species present in the raw material has been observed.

Control measures

Intial level of hazard (H 0 ). See section on raw shellﬁsh.

221 Reduction of hazard (Σ R). The presence of enteric pathogens such as salmonellae, Shigella spp.

FISH AND FISH PRODUCTS

or virus is associated with insufﬁcient thermal processing (lack of control ofΣR). Cooked product should be cooled promptly and held at ≤2 ◦

C. Flotation brines should be refrigerated, and should be changed or treated on a scheduled basis to adequately control microbial loads. At each change, the tank should be cleaned and disinfected. Processing temperatures, cooling rates, and refrigerated/frozen storage temperatures should be monitored on a continuous basis.

Products intended to have an extended refrigerated shelf life must include controls that inhibit growth of psychrotrophic pathogens such as L. monocytogenes and Cl. botulinum (see section on lightly preserved ﬁsh products).

Increase of hazard (Σ I). High numbers of contaminating Staph. aureus are associated with ex- cessive handling by humans and subsequent growth of the organisms. This recontamination (ΣI) is controlled through training of personnel and minimizing direct contact with cooked product (e.g. use of gloves or utensils). This pathogen’s halotolerance may also allow its build up in brines; this can be controlled by refrigeration of the brine and its periodic replacement. Further storage and use of cooked, peeled crustaceans must prevent growth (ΣI) of the bacterium to levels where enterotoxin is formed. Enteric organisms can occur as result of recontamination (ΣI) after thermal processing, often due to improper handling by personnel. Vibrio spp. are most often associated with cross-contamination (ΣI) of cooked product. This can be controlled through proper design of processing facilities, control of the movement of product, personnel and equipment, and adherence to sanitation programs.

Testing. Growth and toxin production is required for Staph. aureus to cause illness and if no prior knowledge of processing (e.g. GHP and HACCP programmes) is available, some assurance of safety can be acquired by microbiological testing. Thus the EU has a microbiological standard for cooked crustaceans for Staph. aureus (EEC, 1991a, 1993) with n = 5, c = 2, m = 100 cfu/g and M = 1000 cfu/g. The EU legislation (EEC, 1991a, 1993) also contains a standard for Salmonella which must be absent in 25 g with n = 5 samples. The legislation also contains standards for either E. coli or thermotolerant coliforms. Cooked crustaceans may be tested for presence of Vibrio spp., which will only appear if recontamination has occurred. If detected, subsequent use of the product should ensure that the organisms are either inactivated (e.g. by re-cooking) or that growth in prevented, e.g. by brining in NaCl–acid-type brines with preservatives.

Spoilage. Cooked crustaceans must be chilled rapidly to control growth of spoilage bacteria. The product is normally frozen and quality deterioration related to protein degradation during frozen storage.

VIII Lightly preserved ﬁsh products

A Introduction Approximately 15% of the world catch of ﬁsh is still processed by curing (drying, salting, or smoking),

often by traditional methods such as air- or sun-drying, hand salting, and smoking over fires. The so- called lightly preserved fish products are products preserved by a combination of preservation parameters such as light salting, cold-smoking, lowering of pH, and cooling. These are products in which the normal Gram-negative spoilage flora is somewhat inhibited and shelf life thus extended as compared to fresh iced fish. The products are not sterile and do allow microbial growth and typically do spoil because of microbial activity. Examples of such products are cold-smoked fish, pickled (“gravad”) fish, roe marinated in soy sauce and brined, and cooked crustaceans in brine.

MICROORGANISMS IN FOODS 6

Smoking of seafood is done principally to produce a food with attractive appearance and ﬂavour. Most smoked seafood are only lightly brined (<6%) and smoked, so they are capable of supporting bacterial growth. Cold-smoked products (<30 ◦

C) typically retain a mixed population of bacteria and depending on storage temperature and atmosphere of packaging different spoilage scenarios prevail. Hot-smoked seafoods (>60 ◦

C) have substantially reduced bacterial populations, but provide an excellent substrate for potential pathogen growth because of elimination of competitive bacteria. Hot-smoked products are categorized as pasteurized products and are discussed below.

Several fish species are used for cold-smoking, but salmon (typically reared salmon) is the most common. The fish are filleted and salted either by dry salting, brining or injection of brine. After a short “freshening” the fillets are cold-smoked. The cold-smoking typically lasts 6–24 h and takes place at 22–30 ◦

C. These lower temperatures are used to avoid coagulation of the fish proteins; thus the fillets retain a raw appearance. After cooling, the fillets are packed or sliced and packed. The shelf life is dependent on the NaCl content but a vacuum-packed product can keep for 3–8 weeks at refrigeration temperatures (Figure 3.9).

Pickled (gravad) fish are typical of the Scandinavian countries and are made from salmon, trout, or halibut. Fish fillets are sprinkled with salt, sugar, and spices (dill) and placed under pressure for 1–3 days at refrigerated temperature (5 ◦ C). The fillets are sliced and the product stored chilled. Shelf life is short; typically 1–2 weeks. This product is different from another type of pickled fish (so-called marinated herring) in which salted or acid brined herring is placed in an acetic acid–NaCl–spice marinade. These products have a much longer shelf life and are described below.

Japanese examples of this category include the marinating of for instance salmon roe in soy sauce followed by packaging and frozen storage. Brined cooked crustaceans are typically made from shrimp or crab meat. The cooked meat is placed in a brine with salt (4–6%), citric acid (pH 4.5–5.5), sugar (or sweetener), and benzoic and sorbic acid. The products are stored chilled and can be stable for 4–6 months.

B Saprophytes and spoilage Curing typically results in a shift from a population dominated by Gram-negative bacteria in the raw

product to one in which Gram-positive organisms dominate. The extent of this shift is related to the severity of the process. Generally species of spore-forming Clostridium and Bacillus, Gram-positive cocci, and lactic acid bacteria are the dominant survivors, but some Gram-negative bacteria (e.g. psychrotrophic Enterobacteriaceae) will also normally survive. During subsequent refrigerated storage, these bacteria will multiply and cause spoilage. In these products, packaging that provides a barrier to oxygen transfer is likely to select for microaerophilic Gram-positive and fermentative Gram-negative species. For example, packages of cold-smoked Canadian and Norwegian salmon stored at 0–2 ◦

C for 80 days had lactobacilli as the predominant spoilage organism (Parisi et al., 1992). The microbial ecology of these products is somewhat similar to packed meat products and minimally processed vegetables

stored in CO 2 atmosphere. Tyramine-producing strains of Carnobacterium piscicola and Lactobacillus viridescens have been isolated from sugar-salted fish during refrigerated storage, and this biogenic amine has been proposed as an index of microbial quality (Leisner et al., 1994). Rapid growth of lactic acid bacteria is seen during chill storage of vacuum-packed cold-smoked fish. The spoilage flora is variable but

typically fall into one of the following three groups: (i) dominated by lactic acid bacteria at ∼10 8 cfu/g, (ii) a combination of lactic acid bacteria (10 7 –10 8 cfu/g), and Enterobacteriaceae (10 6 cfu/g), or (iii) marine vibrios (e.g. P. phosphoreum) often with lactic acid bacteria (Truelstrup Hansen et al. 1998). Spoilage is characterized by development of multiple volatile compounds (Jørgensen et al., 2000) and is caused by Lactobacillus species (curvatus or saké) and some members of the Gram-negative flora. The Lb. curvatus/saké group is capable of degrading sulfur-containing amino acids to hydrogen sulfide.

FISH AND FISH PRODUCTS

Salt (brine, injection, dry salting

Ripen (2-18 h)

Dry (1.5 – 6 h)

Smoke (3-8 h)

Cool / ripen

Chill / freeze

Skin

Slice

Weigh Package

Storage Ship

Figure 3.9 Example of process diagram for production of cold-smoked salmon (Huss et al., 1995).

In contrast, C. piscicola, which often dominates the microﬂora appears to have no adverse effects on sensory quality (Paludan-M¨uller et al., 1998).

Lactic acid bacteria will also become dominant during chill storage of brined crustaceans, although growth is typically very slow. Leuconostoc may cause a ropy slime spoilage if carbohydrates like sucrose are used for sweetening. This may be avoided by using artiﬁcial sweeteners.

C Pathogens Cold-smoked finfish and shellfish as well as other lightly preserved fish products have been implicated

in outbreaks of food poisoning. A hazard analysis of the process/product reveals four major agents: Cl. botulinum type E, L. monocytogenes, histamine, and parasites, assuming that raw materials from areas

MICROORGANISMS IN FOODS 6

with aquatic toxins are not used (IFT, 2001; Huss et al., 1995). However, pathogens from the human- animal reservoir may also be transferred to the products, particularly if manually handled. Hence, in Japan, 69 cases of E. coli O157:H7 were caused by soy sauce marinated salmon roe (Japanese Ministry for Health, Labour and Welfare, 1998).

Clostridium botulinum type E (its spores) may be present on the raw material and will survive the processing. Despite the widespread occurrence of Cl. botulinum in the aquatic environment (Dodds, 1993; Dodds and Austin, 1997), it is not commonly found on smoked ﬁsh. Heinitz and Johnson (1998) failed to detect any spores on 201 commercial vacuum-packed smoked ﬁsh samples.

Neither of the preservation parameters on its own, NaCl or low temperature, can prevent growth and toxin formation in vacuum-packed products. Several studies have evaluated the combinations of NaCl and low temperature required to inhibit growth and toxin formation (Graham et al., 1997). Recently, Dufresne et al. (2000) inoculated cold-smoked trout and found that products stored at 4–12 ◦

C with 1.7% NaCl (water phase salt) spoiled before becoming toxic. At 12 ◦

C, the vacuum-packed trout spoiled in 11–12 days and toxin was detected after 14 days. No toxin was detected at 4 ◦

C for 28 days at which point, the product was considered spoiled. A combination of 3.5% NaCl (WPS) and 5 ◦

C will prevent toxin formation for at least 4 weeks (ACMSF, 1992). Using trout naturally contaminated with Cl. botulinum spores for production of hot-smoked ﬁsh, Cann and Taylor (1979) found no toxin-production in ﬁsh with 3% NaCl stored for 30 days at 10 ◦

C. Finnish studies reported surprisingly rapid toxin production by Cl. botulinum type E inoculated into cold-smoked trout (Hyytiia et al., 1997). Toxin was detected in vacuum-packed cold-smoked trout containing 3.4% NaCl after 4 weeks at 4 ◦

C and after 3 weeks at 8 ◦ C. Oddly, levels of Cl. botulinum appeared to decrease during the same period from 140 to 70–80 cfu/g (Hyytiia et al., 1997). The authors do not discuss the unusual results, but it is possible that the toxin detected did not originate from multiplying cells, but may have been released from cells that lysed.

Recently, there has been a great deal of interest in the potential for growth of L. monocytogenes in cold-smoked seafood and other lightly preserved seafood products. Cold-smoked mussels containing > 10 7 L. monocytogenes /g were implicated as the cause of a cluster of listeriosis cases in Tasmania (Mitchel, 1991; Misrachi et al., 1991). Also, a small out-break of listeriosis in Sweden was traced to cold-smoked and gravad trout (Ericsson et al., 1997). Quantitative risk assessments (FDA/FSIS, 2003) have identiﬁed cold-smoked ﬁsh as a high-risk product with respect to listeriosis.

The pathogen has been isolated from both hot- and cold-smoked finfish (Rørvik et al., 1992; Dillon et al ., 1994) and the processing environment (Jemmi and Keusch, 1994; Fonnesbech Vogel et al., 2001). The incidence in newly processed cold-smoked fish varies from 0% in some smoke houses to 100% in others (Heinitz and Johnson, 1998; Jørgensen and Huss, 1998). A recent US survey (Kraemer, 2001) found 3% of cold-smoked fish (at retail level) to be positive for L. monocytogenes. Hot-smoked fish also was positive at a 3% prevalence and values were identical for imported and domestically produced smoked fish. In an even more recent US survey (Gombas et al. 2003), 4–5% of samples of smoked fish were positive for L. monocytogenes.

The microorganism survives cold-smoking and can subsequently grow to high levels during refrigerated storage, particularly in salmon (Guyer and Jemmi, 1991; Dillon and Patel, 1993; Embarek and Huss, 1993; Peterson et al., 1993; Eklund et al., 1995). It should be emphasized that whilst the bacterium

easily grows to levels of 10 8 cfu/g in inoculated packs (Nilsson et al., 1999), the growth in naturally contaminated products appears to be much slower (lower growth rate) and reach lower maximum numbers (Jørgensen and Huss, 1998). Whilst the vast majority of cold-smoked ﬁsh at retail level only contains low

levels, a few samples were found to contain 10 5 L. monocytogenes per gram (Kraemer, 2001). Testing more than 30 000 ready-to-eat food samples, only two contained levels between 10 4 and 10 6 L. monocytogenes per gram—both being smoked ﬁsh (Gombas et al., 2003). Sanitation and cleanup procedures can eliminate the microorganism; however, recontamination occurs soon after restarting processing (Eklund et al., 1995). Several studies have found that although the raw ﬁsh is probably the original

225 Table 3.16 Number of samples, number of L. monocytogenes positive and randomly ampliﬁed polymorphic DNA (RAPD)

FISH AND FISH PRODUCTS

type of L. monocytogenes of a salmon smoke house

No of samples with RAPD type (2, 3, . . . , X) year

Number of

Raw ﬁsh environment

55 4 1 36 4 1 1 6 2 Smoking environment

Slicing environment (1)

80 3 1 63 1 10 2 Slicing environment (2)

Raw ﬁsh environment

17 3 5 6 3 Smoking environment

Slicing environment (1) 75 9 6 3 Slicing environment (2)

3 2 1 Product

15 6 7 7 X = unique types, isolated only once each (Fonnesbech Vogel et al., 2001).

source of the bacterium, the processing equipment (e.g. slicers) are the most important immediate source of product contamination. In one smoke-house, a particular DNA-type of L. monocytogenes was isolated from the product and from slicing environments over a 4-year period (Fonnesbech Vogel et al., 2001; Table 3.16). Freezing, sodium nitrite and modiﬁed atmosphere packaging, as well as sodium lactate in combination with sodium nitrite and sodium chloride, has been reported to help retard the growth of L. monocytogenes in vacuum packaged salmon (Pelroy et al., 1994a,b). The lack of growth in naturally contaminated products as compared to inoculated trials has been explained by the anti-listerial action of the lactic acid bacteria growing on the product. Thus experiments have shown that growth of L. monocytogenes in cold-smoked salmon may be inhibited by the addition of high levels of carnobac- teria (Nilsson et al., 1999; Duffes et al., 1999).

Histamine and other biogenic amines can be found in gravad and cold-smoked ﬁsh (Leisner et al., 1994; Jørgensen et al., 2000) and concern has been raised particularly while cold-smoking of tuna, which contains high levels of histidine—the precursor for histamine. Although histamine may be formed during chill storage of cold-smoked salmon e.g. by P. phosphoreum, nothing is known about potential histamine formation during the cold-smoking process.

Cold-smoking does not inactivate Anisakis in salmon (Gardiner, 1990) and freezing pre- or post- processing is needed to assure elimination of the parasite. Thus EU legislation prescribes that wild salmon be frozen for at least 24 h at −18 ◦

C at some stage during processing (EEC, 1991b). As discussed (Table 3.6), farmed ﬁsh do not contain parasites.

D CONTROL (cooked crustaceae, frozen or chilled)