l-1

International Journal of

Journal ofFoodMicrobii

ELSEVIER

Food Microbiology 33 (1996) 121-137

Microbiological

spoilage of fish and fish products

Hans Henrik Huss

Lone Gram,

Danish Institute &or Fi&ries Research, Department of Seafood Research, TechnicaE Uniwrsity of Denmark, Building 221, DK-2800 Lyngby, Denmark

Abstract
Spoilage of fresh and lightly preserved fish products is caused by microbial action. This paper reviews the current knowledge in terms of the microbiology of fish and fish products with particular emphasis on identification of specific spoilage bacteria and the qualitative and quantitative biochemical indicators of spoilage. Sizewnnella putrefaciens and Pseu&monas spp. are the specific spoilage bacteria of iced fresh fish regardless of the origin of the fish. Modified atmosphere stored marine fish from temperate waters are spoiled by the CO, resistant Photobacterium phosphoreum whereas Gram-positive bacteria are likely spoilers of CO, packed fish from fresh or tropical waters. Fish products with high salt contents may spoil due to growth of halophilic bacteria (salted fish) or growth of anaerobic bacteria and yeasts (barrel salted fish). Whilst the spoilage of fresh and highly salted fish is well understood, much less is known about spoilage of lightly preserved fish products. It is concluded that the spoilage is probably caused by lactic acid bacteria, certain psychrotrophic EntProhact~riaceue and/or Ph~~~bac~erjt~Fn ~l~~sph~r~~m. However, more work is needed in this area. Ke~#~or& Fish: Fish products; Spoilage association

1. Introduction
Microbiological spoilage of foods may take diverse forms, but all of them are a consequence of microbial growth and/or activity, which manifests itself as changes in the sensory characteristics as shown in Table 1. * Corresponding author. Tel.: + 45 45 252586; fax: f 45 45 884774: e-mail: gram~~f~.min.dk

~l68-1605~96~$15.0~ 8 1996 Elsevier Science B.V. All rights reserved PII SOI 68-I 605(96)01134-8

Raw foods are initially contaminated with a wide variety of ~~icroorganisms, but only a selection of these contaminants is able to colonize the food and grow to high numbers. The term spoilage association has been coined for such a specific microbiaf community. The precise mechanism by which one group of bacteria predominates over another, closely related group is not always fully understood. It is well known that only minor changes in processing and packaging of fish products are causing a dramatic change in the development and composition of the spoilage association and a complete different type of spoilage. However, even for the same type of product, spoilage may develop differently, depending on geographical origin and other unknown factors interacting with the microbial development. The present paper will review the recent findings and try to explain the sensory profile at the time of spoilage in terms of the microbiological activity in the product.

2. Fish as substrate for bacterial growth All food commodities have their own distinctive microbiology. Important factors contributing to the microbiological complexity of seafood are: - specific as well as non-specific contamination of the live animal from the environment and of products during processing; _- growth conditions for microorganisms due to specific intrinsic and extrinsic factors (temperature, u,, pH, Eh, microbial interactions etc.). The wide range of environmental habitats (freshwater to saltwater, tropical waters to arctic waters, pelagic swimmers to bottom dwellers and degree of pollution) and the variety of processing practices (iced fish products to (sterile) canned products) are all important factors in determining the initial contamination of fish and fish products. The part of the microflora which will ultimately grow on the products will be determined by the intrinsic and extrinsic parameters. There are several important specific intrinsic factors in fish which greatly influence the microbiology and spoilage: .._ the poikilotherm nature of the fish and its aquatic environment; - a high post ~~~~rt~~7 pH in the flesh (usually > 6.0); -- the presence of large amounts of Noel-protein-nitrogen (NPN);
Table 1 Microbiological Microbiological

spoilage activity

of foods Sensory manifestation and -flavour

Breakdown of food components Production of extracellular polysdccharide material Growth per se of moulds, bacteria, yeasts
COZ----from carbohydrate Production of diffusible or aminoacids pigments

Production of off-odour Slime formation Large visible pigmented colonies Production of gas Discolouration

or non-pigmented

L. Gram, H.H.

Hun

1 ht.

J. Food Microbiology

33 (1996) 121-137

123

~ the presence of trimethylamine oxide (TMAO) as part of the NPN fraction. Bacteria estabilsh themselves on the outer and inner surfaces of the live fish (gills, skin, gastro-intestinal tract). The poikilotherm nature of fish allows bacteria with a broad temperature range to grow. Thus, the microflora of temperate water fish is dominated by psychrotrophic Gram-negative, rod-shaped bacteria belonging to the genera Pseudomonos, Moraxellu, Acinetobacter, Shewanellu, Fluvobucterium, Vibrionuceue and Aeroemonaduceue, but Gram-positive organisms such as Bacillus, Micrococcus, Clostridium, Luctobacillus and Corynebacterium can also be found in varying proportions. The flora on tropical fish often carries a slightly higher load of Gram-positive and enteric bacteria, but is otherwise similar to the flora on temperate-water fish (Liston, 1980). An important intrinsic factor related to fish flesh is the very high post-mortem pH ( > 6.0). Most fish contain only very little carbohydrate ( < 0.5%) in the muscle tissue and only small amounts of lactic acid are produced post mortem. This has important consequences for the microbiology of fish as amongst other factors it allows the pH sensitive spoilage bacteria Shewanella putrefuciens to grow. The non-protein-nitrogen (NPN) fraction of the fish flesh consists of low-molecular-weight water-soluble nitrogen containing compounds such as free amino acids and nucleotides and is a readily available bacterial growth substrate. The decomposition of the sulphur containing amino acids cysteine and methionine is particularly important in spoilage, as it causes off-odours and -flavours due to formation of hydrogen sulphides and methylmercaptane respectively (Herbert and Shewan, 1975, 1976). Trimethylamineoxide (TMAO) is part of the NPN fraction and its presence in all marine (Hebard et al., 1982) and some fresh water fish (Gram et al., 1989; Anthoni et al., 1990) species is well established. TMAO is known to cause a high (positive) redox potential (Eh) in the fish flesh (Huss and Larsen, 1979, 1980) however, the significance of this is not clear. The spoilage of fresh fish is certainly influenced by the presence of TMAO, particularly under conditions where oxygen is excluded. A number of well defined spoilage bacteria (Shewunellu putrefuciens, Photobacterium phosphoreum, Vibrionaceae) are able to utilize TMAO as the terminal electron acceptor in an anaerobic respiration resulting in off-odours and -flavours due to formation of trimethylamine (TMA) (Gram et al., 1987, 1990; Dalgaard et al., 1993). In sugar-salted herring, the presence of TMAO and the high Eh was established as the protective mechanism against the most common type of spoilage (sweet-sour, rotten putrid) as the organism causing this type spoilage is a strict anaerobe requiring a low Eh for growth (Knochel and Huss, 1984a,b).

3. Principles of bacterial spoilage In its simplest form, food spoilage is a result of microbiological growth per se and becomes evident as visible growth (moulds, pigmented or non-pigmented, slimy bacterial colonies). In such cases, of course, there is a direct relationship between the total numbers of microorganisms and degree of spoilage.

124

10 -

Sensory rejection

60 50

987-

654-

321 0 0 5 10 15 20 25 30 days of storage

Fig. 1. Model of changes in total count (TVC). specilic spoilage organisms (SSO) and chemical indices during chill storage of a fish product (modified from Hubs et al.. 19%). spoilage

IO 0

More often spoilage is as a result of the production of off-odours and -flavours caused by bacterial metabolism. In this case there is no correlation between total numbers of bacteria and spoilage (Caste11 et al., 1948; Huss et al., 1974), since only a fraction of the total fora participates in the spoilage. A clear distinction should be made between the terms spoilage association and spoilage organisms (bacteria) since the first describes merely the bacteria present on the fish when it spoils whereas the latter is the specific group that produces the off-odours and off-flavours associated with spoilage (Fig. 1). It is not an easy task to determine which of the bacteria isolated from the spoiled fish are those causing spoilage, and it requires extensive sensory, microbiological and chemical studies. First, the sensory, microbiological and chemical changes during storage must be studied and quantified, including a determination of the level of a given chemical compound that correlates with spoilage (the chemical spoilage indicator). Second, bacteria are isolated at the point of sensory rejection. Pure and mixed cultures of bacteria are screened in sterile fish substrates for their spoilage potential, i.e., their ability to produce sensory (off-odours) and chemical changes typical of the spoiling product (Caste11 and Anderson, 1948; Herbert et al., 1971; Gram et al., 1987). Spoilage potential can be assessed in substrates such as sterile, raw fish juice (Lerke et al., 196R), heat-sterilized fish juice (Caste11 and Greenough, 1957; Gram et al., 1987; Dalgaard, 1995a) or on sterile muscle blocks (Herbert et al., 1971). The latter is the most complicated, but is also the one which is most comparable to the product.

Finally, the selected strains are tested to evaluate their spoilage activity, i.e., their growth kinetics and their qualitative and quantitative production of off-odours in the product of concern (Gram, 1989; Dalgaard, 1995a). The latter step is important, as some bacteria may produce the chemical compounds associated with spoilage, but are unable to do so in significant amounts at the normal conditions prevailing in a product and they are thus not the specific spoilage bacteria. When stored aerobically, levels of lo-10 cfu/g of specific spoilage bacteria are required to cause spoilage of iced fish. The spoilage of chilled, CO, packed fish is seen at a much lower level of lo7 P. ~~~~~?z~~~~~~ per gram (Dalgaard et al., 1993). This relatively low cell level is due to the large size (5 jlrn) of the bacterium resulting in a high amount of TMA per cell (Dalgaard, 1995a) and thus a higher spoilage activity. Calculating the amount of TMA per cell surface or volume, the production of TMA by P. phosphoreum is, however, not different from that of S. putrefuckm, for example. Some organisms may not be able to produce off-odours in sterile fish substrates but only when the substrate is digested by other members of the spoilage associateion. Therefore, in some products, spoilage potential and acitivity of a particular strain must be evaluated in competition or symbiosis with the normal spoilage association.

4. Spoilage of fresh fish Generally the quality deterioration of fresh fish is characterized sensorically by an initial loss of fresh fish flavour (sweet, seaweedy). After a period where the odour and flavour is described as neutral or non-specific, the first indications of off-odours and flavours are detectable. These will progressively become more pronounced and lead to rejection of the fish. The time to spoilage depends mainly on storage temperature and fish species. The initial quality loss in fish is primarily caused by autolytic changes and is unrelated to microbiological activity. Of particular inlportance in this respect is the degradation of nucleotides (ATP-related compounds) which is caused by autolytic enzymes. It is now widely accepted that the loss of the intermediate nucelotide, inosine monophosphate (IMP), is responsible for the loss of fresh fish flavour, but apart from this, the autolytic changes are contributing to spoilage mainly by making catabolites available for bacterial growth (see Huss, 1995). The breakdown of IMP and inosine proceeds faster in naturally contaminated fish than in sterile samples and it has been shown repeatedly that several bacteria participate in the degradation (see below). The off-odours and -flavours developing in fish stored in air depend on the fish species and origin of the fish. The spoilage of marine temperate-water fish is characterized sensorically by development of offensive fishy, rotten, H,S-off-odours and -flavours. This sensory impression is distinctly different for some tropical fish and freshwater fish, where fruity, sulphydryl off-odours and -flavours are more typical (Lima dos Santos, 1978; Gram et al., 1989).

The spoilage association developing in aerobically stored fish consists typically of Gram-negative psychrotrophic non-fermenting rods. Thus, under aerobic iced storage, the flora is composed almost exclusively of Pseudon7onas sp. and S. putr-&cirn.s. This is true for all fish and shellfish whether caught or harvested in temperate (Levin, 1968; Gram et al., 1987) or sub-tropical and tropical waters (Lannelongue et al., 1982a; Gram et al., 1990: Lima dos Santos, 1978; Shamshad et al., 1990). At ambient temperature (25C), the microflora is dominated by mesophilic Vibrionuwue (Gorczyca and Pek Poh Len, 1985; Gram et al., 1990) and, particularly if the fish are caught in polluted waters, mesophilic EI?tel.ObCI(.t(riCI(.(,C/( (Gram, 1992). Sf1e1var7dlu pwtrcrfilcirrzs is the specific spoilage bacteria of marine temperate-water fish stored aerobically in ice and the number of S. putrclfi~cirns is inversely linearly related to remaining shelf-life of iced cod (Jorgensen et al., 1988). S. pzrrr&c%~.s strains isolated from fish products have similar spoilage potential (Jsrgensen and Huss, 1989: Dalgaard, 1995a), however, the group is phenotypically heterogenous (StenstrGm and Molin, 1990) and on-going work at our laboratory show that there may be a clonal selection during a storage trial (Fonnesbech et al., in preparation). Psruh77orzas sp. are the specific spoilers of iced stored tropical freshwater fish (Lima dos Santos, 1978; Gram et al., 1990) and are also, together with S. putwj&~irns, spoilers of marine tropical fish stored in ice (Gillespie and MacRae. 1975; Gram, 1992). S. putrclfircicns has been isolated from tropical freshwaters. but does not appear to be important in the spoilage of iced freshwater fish from tropical waters (Lima dos Santos. 1978; Gram et al., 1990). This may bc due to occurrence of very low numbers and the inabililty of the organism to compete with high numbers of antagonistic pseudomonads (Gram, 1993; Gram and Melchiorsen. 1996). At ambient temperature, motile aeromonads are the specific spoilers of aerobically stored freshwater fish (Gorczyca and Pek Poh Len, 1985; Gram et al., 1990). Barile et al. (1985) showed that a large proportion of the flora on ambient-stored mackerel consisted of S. pzrtwjkicms, indicating that this bacterium may also take part in the spoilage. In vacuum-packed iced stored fish from temperate marine waters an increased development of TMA is seen (see Fig. 2) while the shelf-life is unaffected compared to aerobically stored fish (Table 2). The number of PLwudomm~s is reduced, but S. putrcfticiem which is capable of anaerobic respiration using TMAO grows to levels of IOh- 10 cfu/g (Gram et al., 1987; Jmgensen et al.. 1988; Dalgaard et al., 1993). Numbers below 10 cfu/g are unlikely to be important in spoilage and consequently other organisms must be involved. Jcrrgensen et al. (1988) observed that vacuumpacked cod contained some very large, almost yeast-like cells and suggested that these were involved in the spoilage. It was recently shown that these cells are heat sensitive Photobucteriunl phosphoreum (Dalgaard et al., 1993). P. phosphoram is a marine vibrio which has escaped microbiologist as it does not grow when pour plating and incubation at high temperatures are used (van Spreekens. 1974; Dalgaard et al., 1993). It is easily isolated from intestines of various fish (van Spreekens, 1974; Dalgaard, 1995a). The organism produces IO- 100 fold more

L. Gram, H.H. Huss / ht. J. Food Microbiology

33 (1996) 121-137

127

70

60 -

1
-

n Vacuum packed v MAP (48% CO,) 0 Aerobic

10

15

20

25

30

days of storage (OC)

Fig. 2. Development modified atmosphere of trimethylamine (48% CO,) (v) (TMA) in cod stored at 0C in air (a), vacuum-packed (modified from Dalgaard et al., 1993). (m) and

TMA per cell than S. putrefaciens (Dalgaard, 1995b) but does not cause off-odours 1977) probably because it does not as foul as S. putrefuciens (van Spreekens, 1977; Dalgaard et al., 1993, Dalgaard, produce volatile sulfides (van Spreekens, 1995a). The spoilage of vacuum-packed fish from temperate marine waters is caused by these two bacteria and differences in initial numbers of S. putrefuciens
Table 2 Effect of packaging Type of product

on the shelf life of chilled

fish and meat products temperature (C)

(Dalgaard,

1995b)

Storage

Shelf life (weeks) Air VP MAP

Meat Beef, pork, poultry Lean fish Cod, pollock, rockfish, trevally Fatty fish Herring. trout, salmon Shellfish Crabs, scampi, scallops Warmwater fish Sheepshead, tilapia, swordfish VP, vacuum-packed; MAP, modified

1.o-4.4 0.064.0 O.OG4.0 0.0&4.0 2.0-4.0 atmosphere packed (High

l-3 l-2 1-2 l/222 l/2--2 CO? concentrations

l-12 l-2 I-2 ~~~ (255100%)).

3321 l-3 l-3 l/2-3 2--4

128

L. Grum, H.H. Huss / Inl. J. Food Microhiolog~~ 33 (1996) 121-137

and P. phosphorrum probably decides which of the two becomes most important. Whilst abundant data exists on marine fish, little is known about the spoilage bacteriology of vacuum-packed freshwater fish. It is unlikely that P. phosphorcum plays a major role in the spoilage of freshwater fish as it is sodium-requiring and Hussain et al. (1976) found that 4 weeks of iced storage of vacuum-packed trout selected for Gram-positive bacteria. CO,-packing of marine fish from temperate waters inhibits the development of the respiratory organisms like Pseudomonas and S. putrcrfitcirns and their numbers rarely exceed 105~10 cfuig. Similar changes resulting in dramatic extensions of shelf-life are seen for meat products (Dainty and Mackey. 1992). However. a number of studies have shown that compared to meat products only limited or no extension of shelf-life is obtained by packing fish in CO, atmosphere (Cann et al., 1983, 1984; Farber. 1991; Dalgaard et al., 1993) (see Table 2). The development of TMA is, as shown in Fig. 2, similar to or delayed only a few days compared to vacuum-packed storage, but increased compared to aerobic storage (Cann et al., 1983; Dalgaard et al., 1993). The presence and subsequent growth of CO, resistant TMA producing P. pho.splzor~urn to levels of 10 10 cfu/g explains these observations. By comparing Table 2 and Fig. 2 it can further be deduced that it is not development and level of TMA per se which causes sensorical rejection of fish, but rather the presence of TMA in combination with other, yet unidentified compounds. Carbon dioxide and vacuum-packing of fish caught in freshwater or warmer waters where these particular heat sensitive, sodium-requiring P. phosphoreun~ are probably not as common, logically result in decrease in TMA production. The microflora becomes dominated by various Gram-positive organisms, mainly lactic acid bacteria (Banks et al., 1980; Lannelongue et al.. 1982a,b; Oberlender et al., 1983; Pedersen and Snabe. 1995). However, as TMA can be detected later in the storage (Reddy et al., 1995; Oberlender et al.. 1983). TMAO-reducing organisms must be present at some level. The specific spoilage bacteria of fresh and packed fish arc summarized in Table 3. Comparison of the chemical compounds developing in naturally spoiling fish and sterile fish has shown that most of the volatile compounds are produced by bacteria (Shewan, 1962). These include TMA, volatile sulphur compounds. aldehydes, ketones, esters. hypoxanthine as well as other low molecular weight compounds. The substrates for the production of volatiles are TMAO, sulphur-containing amino acids, carbohydrates (e.g., lactate and ribose). nucleotides (e.g.. inosine mono-phosphate and inosine) and other NPN molecules. The amino-acids are particularly important substrates for formation of sulphidcs and ammonia (Herbert and Shewan, 1975, 1976; Ringm et al., 1984) (see Table 4). Most bacteria identified as specific spoilage bacteria produce one or several volatile sulphides. S. putw+cicns and some Vitwionacew produce H,S from the sulphur containing amino-acid L-cysteine (Gram et al.. 1987; Stenstriim and Molin, 1990). In contrast, neither Psrudomon~~s nor P. p/msp/~~~w17 produce significant amounts of H,S. Thus, hydrogen sulphide, which is typical of spoiling iced cod stored aerobically, is not detected in spoiling CO, packed cod (Dalgaard et al..

L. Gram, H.H. Huss / Int. J. Food Microbiology 33 (1996) 121-137 Table 3 Specific spoilage Atmosphere

129

bacteria

of fresh and packed organisms

fish stored chilled (<4C) fish Tropical

or in ice (Gram

and Huss, 1996)

Specific spoilage Temperate Marine waters

of fresh, chilled

waters Fresh Pseudomonas spp.

Fresh Pseudomonas sp~.~

Marine S. putrefaciensj Pseudomonas spp. Lactic acid bacteria: others?

aerobic

S. putwfaciens P.reudomonas spp. S. putrt$ciensb! P. phosphoreum h

vacuum

Gram-positive bacteriae Lactic acid bacteria Lactic acid bacteriad

Lactic acid bacteria?

CO?

P. phosphoreum

Lactic acid bacteria; TMAO reducing bacteria

Lactic acid bacteria?/ TMAO reducing bacteria

and Huss (1989). h Dalgaard et al. (1993). Jorgensen et al. (1988). c Dalgaard et al. (1993), Dalgadrd (199Sa). It Assumed to be the most likely spoilage bacteria as typical marine bacteria Hussain et al. (1976). Gillespie and MacRae (1975), Gram (1992). 8 Pedersen and Snabe (1995). h Oberlender et al. (1983), Banks et al. (1980), Lannelongue et al. (1982a). Lima dos Santos (1978) Gram et al. (1990). Reddy et al. (1995).

,s= and,or 1 ?= not known. rl Levin (1968), Herbert et al. (1971), Jergensen

are not present

1993). Methylmercaptan (CH,SH) and dimethylsulphide ((CH,),S) are both formed from methionine (Herbert and Shewan, 1975). Taurine, which is also sulphur-containing, occurs as free amino-acid in very high concentrations in fish muscle and disappears from the fish flesh during storage but this is because of leakage rather than because of bacterial attack (Shewan and Jones, 1957). The development of TMA is in many fish species paralleled by a production of hypoxanthine. Hypoxanthine, which may cause a bitter off-flavour in the fish, can be formed by the autolytic decomposition of nucleotides, but it can also be formed by bacteria; and the rate of bacterial formation is higher than the autolytic. Both Jorgensen et al. (1988) and Dalgaard et al. (1993) showed a linear correlation between the contents of TMA and hypoxanthine during iced storage of packed cod. Several of the spoilage bacteria produce hypoxanthine from inosine or inosine mono-phosphate, including Psetldomonas sp. (Surette et al., 1988) S. putrejkkzs (van Spreekens, 1977; Jorgensen and Huss, 1989; Gram, 1989) and P. phosphoreum (van Spreekens, 1977).

Table 4 Substrate produced by bacteria during storage of fresh and packed fish (Gram and Huss. 1996)

and typical spoilage

compounds

___~... (+) of spoilage compounds

_ Methionine Other Ketones, esters. aldehydes. NH, _ ~~~~~ Hypoxanthine amino acids IMP. inosinc CH,SH. (CH,@ Carbohydrates, lactate Acids

~
Product examples

Production

Substrate:

TMAO

Cysteine

Compounds:

TMA

H,S

+
I

+ _

+ _ _

+ t +

+ + ? ?

+ + _ ?

(+)

(+)

+ +

Lactic acid bacteria Yeast Anaerobic rods

Iced fish Iced lish CO, packed fish Ambient stored fresh fish Lightly preserved fish Lightly preserved fish Sugar-salted fish Sous-vide fish

L. Gram, H.H.

Huss ! Int. J. Food Microbiology

33 (1996) 121-137

131

Contrary to the iced spoilage by S. putrefuciens and the ambient spoilage by Vibrionuceae which is dominated by H,S and TMA, the spoilage caused by Pseudomonas sp. is characterized by absence of these compounds (Gram et al., 1989, 1990). Fruity, rotten, sulphydryl odours and flavours are typical of the Pseudomonus spoilage of iced fish. Pseudomonus sp. produce a number of volatile aldehydes, ketones, esters and sulphides (Edwards et al., 1987; Miller et al., 1973a,b). However, it is not known which specific compounds are responsible for the typical off-odours. The fruity off-odours produced by Pseudomonus fragi originate from monoamino-monocarboxylic amino-acids. Lerke et al. (1967) separated fish juice into a protein and a non-protein fraction and found that the non-protein fraction of a fish juice spoiled as the whole juice whereas only faint off-odours were detected in the protein fraction of the juice. Although some authors have used the number of proteolytic bacteria as indicators of spoilage, it must be concluded that the turnover of the protein fraction is not of major importance in spoilage of fresh fish. Table 4 summarizes what is currently known about the spoilage bacteria on fresh and packed fish and the compounds associated with spoilage.

5. Spoilage

of fish products

5.1. Ligthl?! preserved ,jsh products This group includes fish products preserved by low levels of salt ( < 6% NaCl (w/w) in the water phase) and, for some products, addition of preservatives (sorbate, benzoate, NO, or smoke). Product pH is high ( > 5.0) and they are often packaged under vacuum and must be stored and distributed at chill temperatures ( I SC). This is a group of high-value delicatessen products (cold-smoked, pickeled (gravad) or marinated fish, brined shellfish) that are typically consumed as ready-to-eat products with no heat treatment. The microbial spoilage developing in these products is not well understood. By comparing the sensory changes in naturally contaminated cold-smoked salmon with changes in samples with much reduced bacterial load Truelstrup Hansen et al. (1996) concluded that while autolytic enzymes caused tecture changes (softening) bacterial activity was indeed the cause of spoilage. The off-odours and -flavours developing are variously described as putrid, cabbage-like, sour, bitter, fruity, sweet and the normal shelf-life of this product also varied considerably from about three weeks to eight weeks for vacuum-packed, cold-smoked salmon (445% NaCl in water phase, pH 6.3-6.4) stored at 5C. Several studies have shown that the microflora developing in this type of products is dominated by lactic acid bacteria (LAB) (Magnusson and Traustadottir, 1982; Civera et al., 1995; Truelstrup Hansen et al., 1995; Leisner et al., 1994). Often high levels (lo-10 cfu/g) of LAB are present for several weeks before the product becomes sensorically rejectable, which demonstrates that the often used total count of bacteria is completely useless as a spoilage indicator for this type of products, see Fig. 3.

Truelstrup Hansen (1995) reported that Lactobacillus cmxtus was the most common species occurring, but also Lb. .sukP lbmuricus, Lb. phturum, Curnobwtrrizmzand Lruconostoc sp. were present in smaller numbers. The specific microflora developing seemed to be related to production environment in the smokehouse (an in-house flora). Until recently, it was believed that the lactic acid bacteria were of little importance for the sensory changes during storage of fish products and Leisner (1992) showed that no or very faint off-odours were produced by lactic acid bacteria compared to the very obnoxious off-odours produced by Gr~~rn-neg~~ti~~e spoilers. However, Truelstrup Hansen (1995) found that several lactic acid bacteria were able to produce some of the off-odours (sour, cabbagey, sulphurous) associated with spoilage of cold-smoked salmon. Furthermore, a strain of Lb. saki produced H,S during growth on cold-smoked salmon. Preliminary work at our laboratory has shown that elimination of the lactic acid bacteria by e.g. nisin causes extension of the shelf-life of cold-smoked salmon compared to samples with normal levels of LAB (Gram, Nilsson and Paludan-Moller, unpublished data). Although most studies report dominance of lactic acid bacteria in cold-smoked fish, the chemical changes during storage varies as does the composition of the remaining microbial flora. The TMA content of cold-smoked salmon sometimes increases to 25-30 mg TMA-nitrogen per 100 g (Civera et al., 1995) whereas other
10

9 8 7 6 5 4 3 2 1 0 0 10 20 30 40 50 60 70

days of storage (5C)

Fig. 3. Changes in total count (-s), total psychrotrophic count (Y), lactic acid bacteria (@) and Entevohactrriareac (m) during storage of vacuum-packed cold-smoked salmon (N&l 4.6%) w/w in water phase) at 5C (Truelstrup Hansen et al.. 199s).

L. Grum, H.H. Huss / Inr. J. Food Microbiology 33 (1996) 121-137

133

samples in the same study contained only low levels (5 mg TMA-N per 100 g). Similarly, Truelstrup Hansen et al. (1995) concluded that TMA could not be used as an objective quality indicator of cold-smoked salmon. Since LAB are known not to reduce TMAO to TMA, other bacteria must at times be participating in the spoilage process. Spoiled lightly preserved fish products may also contain low (10 cfu/g) or high ( 106- lo7 cfu/g) levels of Enterobucteriuceae, Brochotrix thermosphnctu, yeasts and Photobacterium phosphoreum (Cann et al., 1984; Civera et al., 1995; Leisner et al., 1994, Truelstrup Hansen et al., 1996). Truelstrup Hansen (1995) concluded that one of three different microfloras were present when cold-smoked salmon was sensoritally rejected: (a) dominated by lactic acid bacteria (107-lo9 cfu/g); (b) dominated by lactic acid bacteria and Enterobucteriuceue (107-lo8 cfu/g); and (c) dominated by Photobacterium phosphoreum ( 106- lo7 cfu/g) with occasional high levels of lactic acid bacteria. The Enterobucteriuceue growing in lightly preserved fish products have been identified as psychrotrophic Hufniu ulvei, Serrutiu Eiquefuciens or Enterobucter sp. All strains produce spoilage off-odours and reduce TMAO but the ability to produce H,S from protein substrates varies (Truelstrup Hansen, 1995) and little is known about their spoilage activity in lightly preserved fish products. 5.2. Suit curing und jkrmentution There are essentially two types of products where the preservative action of salt is the predominant process-dry salted and wet salted or pickeled fish products. Dry-salting is used only for non-fatty fish. There are two types of spoilage of this product. One is growth of the extremely halophilic bacteria which causes a condition known as pink. These pink halophilic bacteria (Hulococcus, Hulobucterium) are strongly proteolytic and produce off-odours and -flavours in the product. The other type of spoilage is moulding by a highly osmophilic type of fungus known as dun (Sporendonemu and Oosporu) (Huss and Valdimarsson, 1990). Wet salting or barrel-salting is used for fatty fish species such as herring and anchovy. The fish are mixed with salt and kept in a closed container. The waterphase salt in barrel-salted herring is typically 15-2033. Three types of spoilage are known for this product (Knochel and Huss, 1984a,b). The most common type is characterized by the presence of sour, sour/sweet and putrid off-odours and -flavours. This type of spoilage is caused by growth of a Gram-negative, halophilic, obligate anerobic rod (up to 10h-107 cfu/g). The growth of this organism is not possible until the general microflora has reduced all the TMAO causing the Eh to drop to negative values. This may take more than one year at chill storage (2-4C) as the general flora consisted of low levels (103-lo5 cfu/g) of mainly Gram-negative, halophilic rods able to reduce TMAO. The second type of spoilage is characterized by the development of fruity off-odours and is caused by growth to levels of 10 cfu/g of osmotolerant yeast species. Finally the term ropiness or ropy brine is used to describe the phenomen that the brine becomes highly viscous or slimy. This third type of spoilage is caused

by a Gram-negative, halophilic, aerobic non-motile rodshaped (Mor~~xell~-like) bacteria (Magnusson and MBller, 198.5). The authors are not aware of any reports of microbiological spoilage of high-salt fermented fish products such as fish sauce and paste. This type of products is. despite their name, not really fermented products as very high salt levels (20-30%) are used and the liquefying action believed to be caused by autolytic enzymes.

Many types of seafood products receive a heat treatment as part of their processing. Particularly the products receiving only a mild heat treatment and distributed at chill temperatures (Refrigerated processed foods with extended durability; REPFEDS) are likely to spoil due to microbial action. UrtUSLidl clostridia have been found as poilers of pasteurized crab meat (Segner, 1992) and Ben Embarek (1994) found that sous vide packed cod stored at 5C spoiled due to growth of a Gram-positive sporeforming bacteria producing extremely obnoxious and putrid off-odours.

6. Conclusions A thorough understanding of the spoilage process and knowledge of the specific spoilage organisms are necessary for design of an optimal, product specific quality assurrance programme or if microbiological data are to be utilized to predict the shelf-life of a product at specific conditions (temperature, packaging). For a number of fish products (heavily preserved as iced or salt cured products) the situation is reasonably well described and understood. One or only a few organisms invariabIy appear as the spoilage organisms in the same type of product under the same geographical conditions. However, the situation in e.g. lightly preserved fish products is much more complicated and the assumption mentioned appears not to hold. Much more work is needed to understand the significance of such factors as the role of bacterial interactions, possible clonal seIection of specially active groups within one species and possible changes of bacteria1 metabolism due to intrisic and extrinsic conditions.

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