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Research ArticleReceived: 2 April 2012 Revised: 20 July 2012 Accepted: 28 July 2012 Published online in Wiley Online Library: 14 September 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5873

Microbiological aspects and shelf lifeof processed seafood productsIoannis S Boziaris,a∗ Anastasios P Stamatioub and George-John E Nychasb

Abstract

BACKGROUND: Fresh fish and seafoods are very perishable products mainly owing to microbial activity of specific spoilagemicro-organisms. Application of hurdle technology leads to a variety of processed products with extended shelf life. In thisstudy, sensory evaluation and microbiological analysis were carried out on 17 processed seafood products stored at 4 ◦C todetermine their shelf life and the predominant spoilage micro-organisms.

RESULTS: Shelf life determined by sensory analysis varied from 66 to 180 days depending on the product. The cause of spoilagefor most of the products was the development of off-flavours/off-odours, while two products were rejected owing to oildiscolouration. Pseudomonads were in most cases below detection limit. H2S-producing bacteria, Brochothrix thermosphactaand Enterobacteriaceae were below detection limit throughout the experiment. The predominant spoilage micro-organismswere lactic acid bacteria and yeasts. Hygiene indicators such as Staphylococcus spp. and total coliforms were also belowdetection limit in all samples.

CONCLUSION: Primarily the initial pH and secondarily the NaCl content determined shelf life duration. Under the appliedconditions, lactic acid bacteria and yeasts predominated. The contribution of chemical oxidation and/or autolysis to spoilageand shelf life might be important for most of the products.c© 2012 Society of Chemical Industry

Keywords: micro-organisms; spoilage; shelf life; processed seafoods

INTRODUCTIONWith the ever growing global need for safe foods to store andtransport from one place to another, an increase in food safetyand shelf life has become necessary. The stability and safety ofmost foods are based on the application of combined hurdlessuch as low temperature, pH and water activity (aw), smoking,thermal treatment, preservatives, etc.1 Fish and seafoods are veryperishable products. Seafoods spoil easily owing to microbial,enzymatic or chemical activities.2 However, fresh fish spoil mainlyowing to microbial action. The high water content and non-proteinnitrogen concentration and relatively high pH of fresh seafoodsrenders them sensitive to microbial attack.3,4

Various preservation treatments are used to extend theshelf life of fish. These treatments inhibit and/or inactivatemicro-organisms. Apart from refrigeration under air or modifiedatmosphere packaging, salting, acidification, drying and smoking(cold or hot) in various combinations can prevent fish spoilageand extend their shelf life.2 The application of such methods leadsto traditional products with exceptional organoleptic propertiesand extended shelf life.5 Microbial populations found in theseproducts are predominantly lactic acid bacteria and yeasts.6

Products that are produced by curing (which includes salting,acidification, fermentation, smoking, preservatives and combina-tions thereof) can be classified as lightly preserved, semi-preservedor heavily salted.6 Lightly preserved fish products have a low saltcontent (<60 g kg−1 NaCl) and a pH above 5.7 These products, e.g.cold-smoked and various marinated products, are usually ready toeat, packed in low oxygen or vacuum and stored at refrigeration

temperatures.3,8 Semi-preserved fish products are preserved byNaCl, organic acids, mostly acetic acid (vinegar), and other preser-vatives. Marinated and salted herring and anchovies are typicalproducts of this category.6

Numerous studies have been published on the spoilage, qualityassessment and shelf life of raw fish caught from northern seas,the Mediterranean Sea and tropical waters. Many studies havealso been carried out on processed fish products preservedby the combined application of salt, acid, smoking, heating,etc., which can be characterised as traditionally preserved fishproducts. However, to our knowledge, there has been no study tocompare microbial spoilage aspects and shelf life in respect of thepreservation hurdles applied.

The aim of this study was to provide data on spoilage and shelflife for a large number of fish products preserved by traditionalmethods and to compare the effects of the hurdles applied on themicrobial spoilage and shelf life of these products.

∗ Correspondence to: Ioannis S Boziaris, Department of Ichthyology and AquaticEnvironment, School of Agricultural Sciences, University of Thessaly, FitokouStreet, GR-38446 Nea Ionia, Volos, Greece. E-mail: [email protected]

a Department of Ichthyology and Aquatic Environment, School of AgriculturalSciences, University of Thessaly, Fitokou Street, GR-38446 Nea Ionia, Volos,Greece

b Laboratory of Microbiology and Biotechnology of Foods, Department of FoodScience and Technology, Agricultural University of Athens, Iera Odos 75,GR-11855 Athens, Greece

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Shelf life of processed seafood products www.soci.org

Table 1. Traditionally preserved fish products used in study and treatments they received. All products were packed in plastic packages filled withsunflower oil

No. Product

Marination(acid g L−1,acidification

duration)Initial pH of

final product

Salting (NaCl solutiong L−1, salting duration,solution/product ratio)

NaCl in finalproduct(g kg−1) Smoking

Thermaltreatment

1 Salted bonito (lakerda) 5.95 Saturated, 5 days, 2 : 1 61

2 Salted anchovies 5.50 Saturated, 7 days, 2 : 1 133

3 Marinated anchovies Acetic 60, 1 h 4.10 160, 1 h, 1 : 1.5 37

4 Marinated sardines Acetic 70, 1.25 h 4.10 60, 1.25 h, 1.5 : 1 19

5 Marinated cod Acetic 70, 1 h 4.85 90, 1 h, 1 : 1 17

6 Marinated herring Acetic 70, 2.5 h 4.48 95, 2.5 h, 1 : 1.5 19

7 Cold-smoked herringfillet with pickledvegetables

5.95 170, 1 h, 2 : 1 27 Cold (27 ◦C)

8 Cold-smoked cod 6.00 200, 45 min, 1 : 1 27 Cold (27 ◦C)

9 Cold-smoked swordfish 5.71 200, 4 h, 2 : 1 47 Cold (27 ◦C)

10 Cold-smoked mackerelfillet

6.21 200, 2 h, 2 : 1 39 Cold (27 ◦C)

11 Cold-smoked mackerelfillet with pepper

5.90 200, 1 h, 2 : 1 30 Cold (27 ◦C)

12 Cold-smoked perch Citric 30, 1 min 5.90 200, 1 h, 1 : 1 27 Cold (27 ◦C)

13 Hot-smoked trout Citric 30, 1 min 6.10 170, 1 h, 2 : 1 29 Hot (68 ◦C, 15 min)

14 Cooked andcold-smoked mackerel

Acetic 60, 1 min 5.50 170, 1 h, 2 : 1 22 Cold (27 ◦C) 80 ◦C, 45 min

15 Cooked and hot-smokedcero (small mackerel)

Acetic 60, 10 s 5.50 200, 2 h, 2 : 1 31 Hot (60 ◦C, 15 min) 80 ◦C, 45 min

16 Cooked marinatedoctopus with greenolives

Acetic 60, 20 min 4.43 30, 25 min, 1 : 1 12 80 ◦C, 2 h

17 Cooked marinatedoctopus with pickledvegetables

Acetic 60, 20 min 4.50 30, 25 min, 1 : 1 13 80 ◦C, 2 h

EXPERIMENTALProcessed seafood productsThe products were manufactured and provided by a leadingseafood industry in Greece. The products, processes and hurdlesapplied (smoking, thermal treatment, pH and NaCl concentration)are shown in Table 1. All products were packed in plastic packageswith sunflower oil. The products were stored at 4 ◦C on arrival atthe laboratory.

Microbiological media and chemicalsAll microbiological media were supplied by Lab M (Heywood,UK), apart from cetrimide/fucidin/cephaloridine (CFC) agar, whichwas supplied by Oxoid (Basingstoke, UK), and streptomycinsulfate/thallous acetate/cycloheximide (actidione) (STAA) agarand Baird–Parker (BP) agar, which were supplied by Biolife ItalianaSrl (Milan, Italy). All chemicals were supplied by Sigma-Aldrich(Steinheim, Germany). Iron agar (IA) was prepared according toGram et al.9 by mixing 20 g L−1 peptone, 3 g L−1 meat extract,3 g L−1 yeast extract, 3 g L−1 ferric citrate, 0.3 g L−1 sodiumthiosulfate, 5 g L−1 NaCl, 0.6 g L−1 L-cysteine and 20 g L−1 agarand adjusting the mixture to pH 7.4.

Microbiological analysisFor microbiological analysis, two packs of each product wereopened and two 10 g samples from each pack were takenaseptically. The 10 g samples were transferred to stomacher bagswith 90 mL of maximum recovery diluent (MRD; 8.5 g L−1 NaCl,

1 g L−1 peptone) and homogenised for 60 s in a stomacher (BugMixer, Interscience, London, UK). Samples of 0.1 mL of the tenfoldserial dilutions were spread in duplicate on the surface of driedmedia in Petri dishes for enumeration of (a) Pseudomonas spp. onCFC agar incubated at 20 ◦C for 48 h, (b) Brochothrix thermosphactaon STAA agar incubated at 25 ◦C for 72 h, (c) yeasts and mouldson rose bengal/chloroamphenicol (RBC) agar incubated at 25 ◦Cfor 72 h and (d) staphylococci on BP agar incubated at 37 ◦C for24 h. Furthermore, 1 mL samples in duplicate were used as pouredplates for enumeration of (a) total viable count (TVC) on platecount agar (PCA) incubated at 20 ◦C for 72 h, (b) H2S-producingbacteria on IA incubated at 20 ◦C for 72 h (counting black coloniesonly), (c) lactic acid bacteria on de Mann–Rogosa–Sharpe (MRS)agar incubated at 25 ◦C for 72 h, (d) Enterobacteriaceae on violetred bile/glucose agar (VRBGA) incubated at 37 ◦C for 24 h and(e) total coliforms on violet red bile agar (VRBA) incubated at 37 ◦Cfor 24 h.

Sensory analysisProduct quality was assessed by ten trained panellists accordingto a methodology described by Dalgaard et al.10 A hedonicscale with three categories was used. Class 1 corresponded tohigh-quality products without any off-odour or off-flavour ordiscolouration, class 2 corresponded to products that had slightoff-odour or off-flavour or discolouration but were still acceptable,and class three (rejection) corresponded to products that hadunacceptable off-odour or off-flavour or discolouration. The shelf

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www.soci.org IS Boziaris, AK Stamatiou, GJE Nychas

Figure 1. Sensory score changes of cold-smoked herring fillet with pickledvegetables during storage at 4 ◦C. Each data point is the mean score of tenpanellists. The vertical line shows the shelf life defined as the point when50% of the panellists (five out of ten) rejected the product.

life was defined as the point when 50% of the panellists (five outof ten) rejected the product.

Statistical analysisMicrobial populations were expressed as mean log colony-formingunits (CFU) g−1 ± standard deviation (SD) of n = 2 × 2 = 4replicates. Cluster analysis was performed using STATISTICA 6.0(StatSoft, Tulsa, OK, USA) between initial pH, salt content and shelflife of the product to determine any relation between these twointrinsic factors and shelf life. Single linkage and Ward’s methodwere used with the Euclidean distances to group individuals basedon the different parameters.

RESULTSTypical graphs of sensory score and microbiological populationchanges during storage of one of the products (cold-smokedherring fillet with pickled vegetables) are shown in Figs 1 and 2respectively. This product was rejected after 81 days of storage at4 ◦C (Fig. 1). At this time point, TVC had reached a level of about 6.2log CFU g−1, while MRS and RBC counts were about 6.0 and 5.3 logCFU g−1 respectively (Fig. 2). The other spoilage bacterial groups,i.e. Pseudomonas spp., H2S-producing bacteria, B. thermosphacta,Enterobacteriaceae, total coliforms and Staphylococcus spp., werebelow detection limit.

Microbial populations at the beginning of storage and theend of shelf life for all products are presented in Table 2. InitialTVCs were low, ranging from below 2 to 3.5 log CFU g−1.TVC increased during storage, in many cases reaching levelshigher than 6 log CFU g−1, apart from marinated anchovies andsardines, where TVC was reduced below the detection limit of10 CFU g−1 (Table 2). H2S-producing bacteria, B. thermosphactaand Enterobacteriaceae populations were below detection limitthroughout the experiment (data not shown). Staphylococcus spp.and total coliforms were not detected initially or during theexperiment (data not shown).

The main spoilage micro-organisms of fresh fish, namelyPseudomonas spp. and H2S-producing bacteria, were in mostcases below detection limit throughout storage (Table 2). The

Figure 2. Microbial population changes (TVC, •; lactic acid bacteria, �;yeasts, �) of cold-smoked herring fillet with pickled vegetables duringstorage at 4 ◦C. Each data point is the mean of four determinations(n = 2 × 2). The vertical line shows the shelf life defined by organolepticevaluation.

predominant spoilage micro-organisms at the end of shelf lifewere lactic acid bacteria and yeasts. The microbial spoilage level(MSL), which is the level of specific spoilage organism (SSO) at thetime of rejection, ranged from 3.9 to 8.2 log CFU g−1 (Table 3).The causes of spoilage were off-flavours/off-odours, apart frommarinated cod and herring, where the products were rejectedowing to oil discolouration (Table 3). The pH of the products didnot change significantly during storage (Table 3).

The results of cluster analysis between initial pH, NaClconcentration and shelf life are shown in Fig. 3. Both singlelinkage and Ward’s method with the Euclidean distances gavethe same product grouping. It can be seen that there are twomain clusters. The first cluster comprises products 3, 4, 15,16 and 17 (marinated anchovies, marinated sardines, cookedand hot-smoked cero, cooked marinated octopus with pickledolives and cooked marinated octopus with pickled vegetablesrespectively). The products of the first cluster have long shelflives (120–180 days), while their initial pH values were below5.5 (Tables 1 and 3). The second cluster comprises the rest ofthe products, with shelf life shorter than 120 days and initialpH higher than 5.5, with the exception of marinated herringand marinated cod, where the initial pH values were 4.48 and4.85 respectively. The second cluster can be divided into twosubclusters. The first subcluster comprises products 1, 2, 9, 10and 11 (salted bonito, salted anchovies, cold-smoked swordfish,cold-smoked mackerel fillet and cold-smoked mackerel fillet withpepper respectively), with shelf life between 90 and 120 daysand salt content greater than 30 g kg−1 (Table 1). The secondsubcluster comprises products 5, 6, 7, 8, 12, 13 and 14 (marinatedcod, marinated herring, cold-smoked herring fillet with pickledvegetables, cold-smoked cod, cold-smoked perch, hot-smokedtrout and cooked and cold-smoked mackerel respectively). Theseproducts have a short shelf life (66–75 days) and a salt contentless than 30 g kg−1 (Tables 1 and 3).

The initial pH of the product seems to play a primary role inshelf life. Indeed, products of the first cluster had exceptionallylong shelf lives. Marinated products with initial pH as low as 4.10,such as marinated anchovies and marinated sardines, had a shelflife of up to 180 days (Table 3). In these two products the low pH

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Table 2. Initial and final microbial populations (mean log CFU g−1 ± SD of n = 4 replicates) of traditionally preserved fish products stored at 4 ◦C

TVC Lactic acid bacteria Yeasts Pseudomonas spp.

No. Product Initial Final Initial Final Initial Final Initial Final

1 Salted bonito (lakerda) 3.2 ± 0.1 4.4 ± 0.2 <1 <1 <2 4.3 ± 0.2 <2 2.7 ± 0.1

2 Salted anchovies 3.3 ± 0.1 4.3 ± 0.3 <1 <1 2.8 ± 0.2 3.9 ± 0.2 2.9 ± 0.1 <2

3 Marinated anchovies <1 <1 <1 <1 <2 <2 <2 <2

4 Marinated sardines 2.9 ± 0.2 <1 <1 <1 <2 <2 <2 <2

5 Marinated cod 2.7 ± 0.1 7.9 ± 0.4 2.6 ± 0.1 7.7 ± 0.4 3.2 ± 0.2 5.9 ± 0.2 <2 <2

6 Marinated herring 2.7 ± 0.2 5.3 ± 0.3 2.7 ± 0.2 5.0 ± 0.2 2.8 ± 0.1 5.1 ± 0.1 <2 <2

7 Cold-smoked herring fillet with pickled vegetables 3.3 ± 0.2 6.2 ± 0.2 2.6 ± 0.1 6.0 ± 0.3 3.0 ± 0.2 5.3 ± 0.3 <2 <2

8 Cold-smoked cod 2.3 ± 0.2 6.5 ± 0.3 <1 6.2 ± 0.2 <2 5.4 ± 0.2 <2 <2

9 Cold-smoked swordfish 2.3 ± 0.2 5.0 ± 0.1 <1 3.3 ± 0.2 <2 4.9 ± 0.2 <2 2.3 ± 0.3

10 Cold-smoked mackerel fillet 2.8 ± 0.2 5.4 ± 0.2 <1 <1 2.3 ± 0.2 5.0 ± 0.2 2.5 ± 0.2 3.1 ± 0.1

11 Cold-smoked mackerel fillet with pepper 3.5 ± 0.4 5.7 ± 0.3 2.9 ± 0.2 5.5 ± 0.3 <2 <2 <2 <2

12 Cold-smoked perch 3.2 ± 0.3 8.2 ± 0.3 2.5 ± 0.1 8.2 ± 0.5 2.3 ± 0.3 5.0 ± 0.2 <2 <2

13 Hot-smoked trout <1 7.3 ± 0.3 <1 6.8 ± 0.2 <2 <2 <2 <2

14 Cooked and cold-smoked mackerel <1 8.0 ± 0.4 <1 7.4 ± 0.3 2.3 ± 0.2 3.6 ± 0.2 <2 3.3 ± 0.2

15 Cooked and hot-smoked cero (small mackerel) 3.1 ± 0.2 4.8 ± 0.2 <1 <1 <2 4.9 ± 0.3 2.4 ± 0.2 2.7 ± 0.2

16 Cooked marinated octopus with green olives 2.5 ± 0.1 4.2 ± 0.3 <1 <1 2.5 ± 0.2 4.1 ± 0.2 <2 <2

17 Cooked marinated octopus with pickled vegetables 3.2 ± 0.1 5.0 ± 0.2 <1 <1 3.0 ± 0.1 5.0 ± 0.3 <2 <2

value of 4.10 caused the microbial populations to decline belowdetection limit (Table 3). Products with initial pH between 4.4and 5.5, such as cooked and marinated octopus and cooked andhot-smoked cero, had a shelf life of 150 days (Table 3). In theseproducts, microbial growth was inhibited, presumably owing tothe low pH. Indeed, the MSL was below 105 CFU g−1 (Table 3).Nevertheless, in marinated herring and marinated cod, despitetheir low pH values of 4.48 and 4.85 respectively, shelf life was nohigher than 70 days (Table 3). In these two products, yeast andlactic acid bacteria managed to grow, reaching population levelsas high as 7.9 and 5.3 log CFU g−1 in marinated cod and marinated

herring respectively (Table 3). However, the cause of spoilage inthese two products was oil discolouration, which might be due tochemical oxidation of lipids rather than microbial metabolites.

Salt content seems to determine the shelf life of products withinitial pH above 5.5 (products of the second cluster). Products withhigh salt content, such as salted bonito (61 g kg−1 NaCl) and saltedanchovies (133 g kg−1 NaCl), and all cold-smoked products withmore than 30 g kg−1 NaCl (cold-smoked swordfish, cold-smokedmackerel fillet and cold-smoked mackerel fillet with pepper) had ashelf life ranging from 90 to 120 days (Table 3). In salted anchovies,salted bonito, cold-smoked swordfish and cold-smoked mackerel

Table 3. Shelf life duration, cause of spoilage, specific spoilage organism (SSO), microbial spoilage level (MSL) and pH change of traditionallypreserved fish products stored at 4 ◦C

No. ProductShelf life

(days) Cause of spoilage

Predominantmicro-organism

(SSO)MSL (logCFU g−1) pH change

1 Salted bonito (lakerda) 103 Off-odour/off-flavour Yeasts 4.3 −0.04

2 Salted anchovies 121 Off-odour/off-flavour Yeasts 3.9 +0.01

3 Marinated anchovies 180 Off-odour/off-flavour – – −0.05

4 Marinated sardines 170 Off-odour/off-flavour – – −0.10

5 Marinated cod 66 Oil discolouration LAB 7.7 −0.23

6 Marinated herring 70 Oil discolouration LAB/yeasts 5.0/5.1 +0.07

7 Cold-smoked herring fillet with pickled vegetables 81 Off-odour/off-flavour LAB 6.0 −0.75

8 Cold-smoked cod 73 Off-odour/off-flavour LAB 6.2 −0.31

9 Cold-smoked swordfish 110 Off-odour/off-flavour Yeasts 4.9 −0.20

10 Cold-smoked mackerel fillet 102 Off-odour/off-flavour Yeasts 5.0 −0.11

11 Cold-smoked mackerel fillet with pepper 102 Off-odour/off-flavour LAB 5.5 −0.02

12 Cold-smoked perch 73 Off-odour/off-flavour LAB 8.2 −0.12

13 Hot-smoked trout 73 Off-odour/off-flavour LAB 6.8 −0.30

14 Cooked and cold-smoked mackerel 75 Off-odour/off-flavour LAB 7.4 +0.45

15 Cooked and hot-smoked cero (small mackerel) 150 Off-odour/off-flavour Yeasts 4.9 −0.03

16 Cooked marinated octopus with green olives 150 Off-odour/off-flavour Yeasts 4.1 −0.02

17 Cooked marinated octopus with pickled vegetables 150 Off-odour/off-flavour Yeasts 5.0 −0.13


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Figure 3. Cluster plot using single linkage and Euclidean distances of 17 products according to initial pH, salt content and shelf life.

fillet the SSOs were yeasts, while in cold-smoked mackerel filletwith pepper the SSOs were lactic acid bacteria. The MSL was below105.5 CFU g−1 in all cases (Table 3).

Finally, products with less than 30 g kg−1 NaCl, such as cold-smoked cod, cold-smoked perch, cold-smoked herring fillet,hot-smoked trout and cooked and cold-smoked mackerel, hada shelf life ranging from 73 to 81 days (Table 3). Lactic acid bacteriawere the SSOs with MSL above 106 CFU g−1 in all cases (Table 3).These products were lightly preserved, allowing micro-organismsto grow and reach levels that can cause microbial spoilage.

DISCUSSIONThe differences found in moisture and NaCl contents, aw, pH,smoking intensity, etc. of processed fish products definitelyaffect their shelf life.11 Shelf lives higher than 150 days havebeen reported for products such as marinated sardines12 andsalted anchovies,13 while for other traditional products such assalted bonito (lakerda) the shelf life was about 90 days.14 Lightlypreserved products such as cold-smoked salmon had a shelflife shorter than 50 days.15 – 17 The shelf life of cold-smoked fishvaries with species, degree of smoking and salt content.16,17

Smoking and/or heat treatment did not affect the shelf life of ourproducts, at least not to the extent that pH and salt content did. Itseems that these processes, especially heating and hot smoking,enhance the reduction in initial microbial load but do not affectthe growth of the remaining microbial populations, which areinhibited by the intrinsic properties (pH and NaCl content) only.Indeed, Kilinc and Cackli18 found that pasteurisation did notaffect the shelf life of sardine marinades stored at 4 ◦C, whileincreased salt content extended the shelf life of cold-smokedsalmon.17

Traditionally, processed seafoods are ready-to-eat products,so microbiological safety is of great concern. Staphylococciand enteric pathogens such as salmonellae might contaminate

the products as a result of poor hygiene conditions, cross-contamination from personnel and other sources, etc. Salmonellaand Staphylococcus aureus can survive for long time in seafoodswith low aw.19,20 Enterobacteriaceae and S. aureus can alsowithstand low pH for quite a long time during storage of marinatedPacific saury.7 The very low populations of total coliforms andS. aureus (below detection limit) found in our products confirm thegood hygiene conditions during manufacturing and imply thatthe possibility of the presence of enteric pathogens is very low.However, it is well established that pathogenic micro-organismscan adapt and develop resistance to hurdles such as low aw

and pH,21 and ready-to-eat seafood products have also beenassociated with listeriosis outbreaks.22 To document the safety ofsuch products, a different experimental design is required, but thiswas not among the aims of the present work.

The selection of spoilage microbiota was affected by low pHand NaCl content. Spoilage of fresh fish is caused by metabolicactivity of micro-organisms such as Pseudomonas spp. andShewanella putrefaciens.3 However, in our products the SSOswere lactic acid bacteria and yeasts. It is widely accepted thathurdles such as low pH and aw inhibit the spoilage Gram-negative microbiota, while they favour the growth of lactic acidbacteria and yeasts.23 As the intensity of preservation hurdlesis increased compared with fresh fish, changes from Gram-negative bacteria to lactic acid bacteria and yeasts have beenrecorded.24 Lactobacillus spp. were the predominant microbiotain marinated anchovies, sardines and pacific saury,7,18,25 andLactobacillus alimentarius was the SSO in marinated herring.26

Lactic acid bacteria were the main spoilage microbiota of saltedanchovies,27 cold-smoked trout28 and salmon,17,29 while yeastswere found in salted bonito.14 Other micro-organisms havealso been reported as part of the spoilage microbiota, such asEnterobacteriaceae, staphylococci and halophilic pediococci insalted anchovies.27,30 Enterobacteriaceae and B. thermosphactatogether with Lactobacillus spp. and yeasts have been reported in


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cold-smoked salmon,15,17,29 while Micrococcus spp. together withLactobacillus spp. have been found in marinated anchovies.25

It can be seen from the present study that the populationlevel of dominant spoilage micro-organisms at the end of shelflife was dependent on the intensity of the hurdles applied. Inproducts with low pH or high NaCl content, microbial populationshardly reach 106 CFU g−1. The marination process and itsintensity greatly affect the survival and growth of microbialpopulations. Populations usually drop below detection limit duringmarination,31 while the spoilage population can range from 105

to 102 CFU g−1.7,18,32,33 As an exception, acid-resistant spoilagepopulations as high as 107 and 109 CFU g−1 were reportedin marinated and acetic acid-preserved herring respectively.26,34

Salts and brines also suppress the maximum population density ofspoilage bacteria. In traditional salted anchovies, total mesophilicand lactic acid bacteria reached populations of about 106 and103.5 CFU g−1 respectively,27 while in brined anchovies themicrobial population level was affected by brine concentration:the higher the brine concentration, the lower the microbialspoilage population.13 In cold-smoked salmon, which is a lightlyprocessed seafood, populations of about 107 –108 CFU g−1 havebeen reported.15 – 17,29

Spoilage of fresh fish is caused by metabolic activity ofmicro-organisms such as Pseudomonas spp., S. putrefaciens,Photobacterium phosphoreum, etc., with an MSL usually above107 CFU g−1.3,4 The lower MSL in most of our samples impliesthat mechanisms other than microbial activity might contributeto the quality deterioration. Despite microbial activity being themain cause of spoilage of lightly preserved products such as cold-smoked salmon, autolytic changes can also play a role.35 Variouschemical reactions such as lipid oxidation may contribute to thespoilage of processed fish products.2,3 The thiobarbituric acid(TBA) value was increased in marinated sardines and shrimps,18,33

while a slight increase was observed in brined anchovies13 andhot-smoked tilapia.36 The application of hurdles such as lowpH and aw, smoking, etc. primarily inhibits microbial growth,while it does not affect the rate of chemical oxidation of lipids.Production of total volatile basic nitrogen in salted and smokedmackerel was inhibited, in contrast to the TBA value.37 Yanaret al.36 also found that oxidative rancidity increased as a resultof increasing salt content. It seems that mechanisms otherthan microbial activity, such as autolytic changes and chemicaloxidation, might contribute to the quality deterioration of suchproducts.

CONCLUSIONSDespite the fact that the shelf life of traditionally processed fishproducts varies with moisture content, NaCl concentration, pH, aw

and smoking and heating intensity, it seems that primarily the pHand secondarily the NaCl content determined the shelf life durationof our products. The spoilage microbiota was dominated by lacticacid bacteria and yeasts. The higher the intensity of preservationhurdles, the lower is the contribution of microbial activity tospoilage mechanisms and shelf life duration. The contribution ofchemical oxidation and autolysis to spoilage and shelf life shouldbe studied in future work.

ACKNOWLEDGEMENTWe would like to thank NIREAS SA, Greece for the provision ofseafood products.

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