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Research Article
Received: 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 life
of processed seafood products
Ioannis S Boziaris,
a
Anastasios P Stamatiou
b
and George-John E Nychas
b
Abstract
BACKGROUND: Fresh sh and seafoods are very perishable products mainly owing to microbial activity of specic spoilage
micro-organisms. Application of hurdle technology leads to a variety of processed products with extended shelf life. In this
study, sensory evaluation and microbiological analysis were carried out on 17 processed seafood products stored at 4

C to
determine their shelf life and the predominant spoilage micro-organisms.
RESULTS: Shelf life determined by sensory analysis varied from66 to 180 days depending on the product. The cause of spoilage
for most of the products was the development of off-avours/off-odours, while two products were rejected owing to oil
discolouration. Pseudomonads were in most cases below detection limit. H
2
S-producing bacteria, Brochothrix thermosphacta
and Enterobacteriaceae were below detection limit throughout the experiment. The predominant spoilage micro-organisms
were lactic acid bacteria and yeasts. Hygiene indicators such as Staphylococcus spp. and total coliforms were also below
detection limit in all samples.
CONCLUSION: Primarily the initial pH and secondarily the NaCl content determined shelf life duration. Under the applied
conditions, lactic acid bacteria and yeasts predominated. The contribution of chemical oxidation and/or autolysis to spoilage
and shelf life might be important for most of the products.
c 2012 Society of Chemical Industry
Keywords: micro-organisms; spoilage; shelf life; processed seafoods
INTRODUCTION
With the ever growing global need for safe foods to store and
transport from one place to another, an increase in food safety
and shelf life has become necessary. The stability and safety of
most foods are based on the application of combined hurdles
such as low temperature, pH and water activity (a
w
), smoking,
thermal treatment, preservatives, etc.
1
Fish and seafoods are very
perishable products. Seafoods spoil easily owing to microbial,
enzymatic or chemical activities.
2
However, fresh sh spoil mainly
owingtomicrobial action. Thehighwater content andnon-protein
nitrogen concentration and relatively high pH of fresh seafoods
renders them sensitive to microbial attack.
3,4
Various preservation treatments are used to extend the
shelf life of sh. These treatments inhibit and/or inactivate
micro-organisms. Apart from refrigeration under air or modied
atmosphere packaging, salting, acidication, drying and smoking
(cold or hot) in various combinations can prevent sh spoilage
and extend their shelf life.
2
The application of such methods leads
to traditional products with exceptional organoleptic properties
and extended shelf life.
5
Microbial populations found in these
products are predominantly lactic acid bacteria and yeasts.
6
Products that are produced by curing (which includes salting,
acidication, fermentation, smoking, preservatives and combina-
tions thereof) canbeclassiedas lightly preserved, semi-preserved
or heavily salted.
6
Lightly preserved sh products have a low salt
content (<60 g kg
1
NaCl) anda pHabove 5.
7
These products, e.g.
cold-smoked and various marinated products, are usually ready to
eat, packed in low oxygen or vacuum and stored at refrigeration
temperatures.
3,8
Semi-preserved sh products are preserved by
NaCl, organic acids, mostly acetic acid (vinegar), and other preser-
vatives. Marinated and salted herring and anchovies are typical
products of this category.
6
Numerous studies have been published on the spoilage, quality
assessment and shelf life of raw sh caught from northern seas,
the Mediterranean Sea and tropical waters. Many studies have
also been carried out on processed sh products preserved
by the combined application of salt, acid, smoking, heating,
etc., which can be characterised as traditionally preserved sh
products. However, to our knowledge, there has been no study to
compare microbial spoilage aspects and shelf life in respect of the
preservation hurdles applied.
The aim of this study was to provide data on spoilage and shelf
life for a large number of sh products preserved by traditional
methods and to compare the effects of the hurdles applied on the
microbial spoilage and shelf life of these products.

Correspondence to: Ioannis S Boziaris, Department of Ichthyology and Aquatic

Environment, School of Agricultural Sciences, University of Thessaly, Fitokou
Street, GR-38446 Nea Ionia, Volos, Greece. E-mail: boziaris@uth.gr
a Department of Ichthyology and Aquatic Environment, School of Agricultural
Sciences, University of Thessaly, Fitokou Street, GR-38446 Nea Ionia, Volos,
Greece
b Laboratory of Microbiology and Biotechnology of Foods, Department of Food
Science and Technology, Agricultural University of Athens, Iera Odos 75,
GR-11855 Athens, Greece
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Table 1. Traditionally preserved sh products used in study and treatments they received. All products were packed in plastic packages lled with
sunower oil
No. Product
Marination
(acid g L
1
,
acidication
duration)
Initial pH of
nal product
Salting (NaCl solution
g L
1
, salting duration,
solution/product ratio)
NaCl in nal
product
(g kg
1
) Smoking
Thermal
treatment
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 herring
llet with pickled
vegetables
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 swordsh 5.71 200, 4 h, 2 : 1 47 Cold (27

C)
10 Cold-smoked mackerel
llet
6.21 200, 2 h, 2 : 1 39 Cold (27

C)
11 Cold-smoked mackerel
llet 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 and
cold-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-smoked
cero (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 marinated
octopus with green
olives
Acetic 60, 20 min 4.43 30, 25 min, 1 : 1 12 80

C, 2 h
17 Cooked marinated
octopus with pickled
vegetables
Acetic 60, 20 min 4.50 30, 25 min, 1 : 1 13 80

C, 2 h
EXPERIMENTAL
Processed seafood products
The products were manufactured and provided by a leading
seafood industry in Greece. The products, processes and hurdles
applied (smoking, thermal treatment, pHand NaCl concentration)
are shown in Table 1. All products were packed in plastic packages
with sunower oil. The products were stored at 4

C on arrival at
the laboratory.
Microbiological media and chemicals
All microbiological media were supplied by Lab M (Heywood,
UK), apart fromcetrimide/fucidin/cephaloridine (CFC) agar, which
was supplied by Oxoid (Basingstoke, UK), and streptomycin
sulfate/thallous acetate/cycloheximide (actidione) (STAA) agar
and BairdParker (BP) agar, which were supplied by Biolife Italiana
Srl (Milan, Italy). All chemicals were supplied by Sigma-Aldrich
(Steinheim, Germany). Iron agar (IA) was prepared according to
Gram 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
sodium
thiosulfate, 5 g L
1
NaCl, 0.6 g L
1
L-cysteine and 20 g L
1
agar
and adjusting the mixture to pH 7.4.
Microbiological analysis
For microbiological analysis, two packs of each product were
opened and two 10 g samples from each pack were taken
aseptically. The 10 g samples were transferred to stomacher bags
with 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 (Bug
Mixer, Interscience, London, UK). Samples of 0.1 mL of the tenfold
serial dilutions were spread in duplicate on the surface of dried
media in Petri dishes for enumeration of (a) Pseudomonas spp. on
CFCagar incubatedat 20

Cfor 48 h, (b) Brochothrixthermosphacta

on STAA agar incubated at 25

C for 72 h, (c) yeasts and moulds

on rose bengal/chloroamphenicol (RBC) agar incubated at 25

C
for 72 h and (d) staphylococci on BP agar incubated at 37

C for
24 h. Furthermore, 1 mL samples in duplicate were used as poured
plates for enumeration of (a) total viable count (TVC) on plate
count agar (PCA) incubated at 20

C for 72 h, (b) H
2
S-producing
bacteria on IA incubated at 20

C for 72 h (counting black colonies

only), (c) lactic acid bacteria on de MannRogosaSharpe (MRS)
agar incubated at 25

C for 72 h, (d) Enterobacteriaceae on violet

red bile/glucose agar (VRBGA) incubated at 37

C for 24 h and
(e) total coliforms on violet red bile agar (VRBA) incubated at 37

C
for 24 h.
Sensory analysis
Product quality was assessed by ten trained panellists according
to a methodology described by Dalgaard et al.
10
A hedonic
scale with three categories was used. Class 1 corresponded to
high-quality products without any off-odour or off-avour or
discolouration, class 2 corresponded to products that had slight
off-odour or off-avour or discolouration but were still acceptable,
and class three (rejection) corresponded to products that had
unacceptable off-odour or off-avour or discolouration. The shelf
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Figure 1. Sensory score changes of cold-smoked herring llet with pickled
vegetables during storage at 4

C. Each data point is the mean score of ten

panellists. The vertical line shows the shelf life dened as the point when
50% of the panellists (ve out of ten) rejected the product.
life was dened as the point when 50% of the panellists (ve out
of ten) rejected the product.
Statistical analysis
Microbial populations wereexpressedas meanlogcolony-forming
units (CFU) g
1
standard deviation (SD) of n = 2 2 = 4
replicates. Cluster analysis was performed using STATISTICA 6.0
(StatSoft, Tulsa, OK, USA) between initial pH, salt content and shelf
life of the product to determine any relation between these two
intrinsic factors and shelf life. Single linkage and Wards method
were usedwiththe Euclideandistances togroupindividuals based
on the different parameters.
RESULTS
Typical graphs of sensory score and microbiological population
changes during storage of one of the products (cold-smoked
herring llet with pickled vegetables) are shown in Figs 1 and 2
respectively. This product was rejected after 81 days of storage at
4

C(Fig. 1). At this time point, TVChadreacheda level of about 6.2

log CFUg
1
, while MRS and RBCcounts were about 6.0 and 5.3 log
CFU g
1
respectively (Fig. 2). The other spoilage bacterial groups,
i.e. Pseudomonas spp., H
2
S-producing bacteria, B. thermosphacta,
Enterobacteriaceae, total coliforms and Staphylococcus spp., were
below detection limit.
Microbial populations at the beginning of storage and the
end of shelf life for all products are presented in Table 2. Initial
TVCs were low, ranging from below 2 to 3.5 log CFU g
1
.
TVC increased during storage, in many cases reaching levels
higher than 6 log CFU g
1
, apart from marinated anchovies and
sardines, where TVC was reduced below the detection limit of
10 CFU g
1
(Table 2). H
2
S-producing bacteria, B. thermosphacta
and Enterobacteriaceae populations were below detection limit
throughout the experiment (data not shown). Staphylococcus spp.
and total coliforms were not detected initially or during the
experiment (data not shown).
The main spoilage micro-organisms of fresh sh, namely
Pseudomonas spp. and H
2
S-producing bacteria, were in most
cases below detection limit throughout storage (Table 2). The
Figure 2. Microbial population changes (TVC, ; lactic acid bacteria, ;
yeasts, ) of cold-smoked herring llet with pickled vegetables during
storage at 4

C. Each data point is the mean of four determinations

(n = 2 2). The vertical line shows the shelf life dened by organoleptic
evaluation.
predominant spoilage micro-organisms at the end of shelf life
were lactic acid bacteria and yeasts. The microbial spoilage level
(MSL), which is the level of specic spoilage organism (SSO) at the
time of rejection, ranged from 3.9 to 8.2 log CFU g
1
(Table 3).
The causes of spoilage were off-avours/off-odours, apart from
marinated cod and herring, where the products were rejected
owing to oil discolouration (Table 3). The pH of the products did
not change signicantly during storage (Table 3).
The results of cluster analysis between initial pH, NaCl
concentration and shelf life are shown in Fig. 3. Both single
linkage and Wards method with the Euclidean distances gave
the same product grouping. It can be seen that there are two
main clusters. The rst cluster comprises products 3, 4, 15,
16 and 17 (marinated anchovies, marinated sardines, cooked
and hot-smoked cero, cooked marinated octopus with pickled
olives and cooked marinated octopus with pickled vegetables
respectively). The products of the rst cluster have long shelf
lives (120180 days), while their initial pH values were below
5.5 (Tables 1 and 3). The second cluster comprises the rest of
the products, with shelf life shorter than 120 days and initial
pH higher than 5.5, with the exception of marinated herring
and marinated cod, where the initial pH values were 4.48 and
4.85 respectively. The second cluster can be divided into two
subclusters. The rst subcluster comprises products 1, 2, 9, 10
and 11 (salted bonito, salted anchovies, cold-smoked swordsh,
cold-smoked mackerel llet and cold-smoked mackerel llet with
pepper respectively), with shelf life between 90 and 120 days
and salt content greater than 30 g kg
1
(Table 1). The second
subcluster comprises products 5, 6, 7, 8, 12, 13 and 14 (marinated
cod, marinated herring, cold-smoked herring llet with pickled
vegetables, cold-smoked cod, cold-smoked perch, hot-smoked
trout and cooked and cold-smoked mackerel respectively). These
products have a short shelf life (6675 days) and a salt content
less than 30 g kg
1
(Tables 1 and 3).
The initial pH of the product seems to play a primary role in
shelf life. Indeed, products of the rst cluster had exceptionally
long shelf lives. Marinated products with initial pH as low as 4.10,
such as marinated anchovies and marinated sardines, had a shelf
life of up to 180 days (Table 3). In these two products the low pH
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Table 2. Initial and nal microbial populations (mean log CFU g
1
SD of n = 4 replicates) of traditionally preserved sh 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 llet 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 swordsh 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 llet 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 llet 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 below
detection limit (Table 3). Products with initial pH between 4.4
and 5.5, such as cooked and marinated octopus and cooked and
hot-smoked cero, had a shelf life of 150 days (Table 3). In these
products, microbial growth was inhibited, presumably owing to
the low pH. Indeed, the MSL was below 10
5
CFU g
1
(Table 3).
Nevertheless, in marinated herring and marinated cod, despite
their low pH values of 4.48 and 4.85 respectively, shelf life was no
higher than 70 days (Table 3). In these two products, yeast and
lactic acid bacteria managed to grow, reaching population levels
as high as 7.9 and 5.3 log CFUg
1
in marinated cod and marinated
herring respectively (Table 3). However, the cause of spoilage in
these two products was oil discolouration, which might be due to
chemical oxidation of lipids rather than microbial metabolites.
Salt content seems to determine the shelf life of products with
initial pHabove 5.5 (products of the second cluster). Products with
highsalt content, suchas saltedbonito(61 g kg
1
NaCl) andsalted
anchovies (133 g kg
1
NaCl), and all cold-smoked products with
more than 30 g kg
1
NaCl (cold-smoked swordsh, cold-smoked
mackerel llet and cold-smoked mackerel llet with pepper) had a
shelf life rangingfrom90 to120 days (Table 3). Insaltedanchovies,
salted bonito, cold-smoked swordsh and cold-smoked mackerel
Table 3. Shelf life duration, cause of spoilage, specic spoilage organism (SSO), microbial spoilage level (MSL) and pH change of traditionally
preserved sh products stored at 4

C
No. Product
Shelf life
(days) Cause of spoilage
Predominant
micro-organism
(SSO)
MSL (log
CFU g
1
) pH change
1 Salted bonito (lakerda) 103 Off-odour/off-avour Yeasts 4.3 0.04
2 Salted anchovies 121 Off-odour/off-avour Yeasts 3.9 +0.01
3 Marinated anchovies 180 Off-odour/off-avour 0.05
4 Marinated sardines 170 Off-odour/off-avour 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 llet with pickled vegetables 81 Off-odour/off-avour LAB 6.0 0.75
8 Cold-smoked cod 73 Off-odour/off-avour LAB 6.2 0.31
9 Cold-smoked swordsh 110 Off-odour/off-avour Yeasts 4.9 0.20
10 Cold-smoked mackerel llet 102 Off-odour/off-avour Yeasts 5.0 0.11
11 Cold-smoked mackerel llet with pepper 102 Off-odour/off-avour LAB 5.5 0.02
12 Cold-smoked perch 73 Off-odour/off-avour LAB 8.2 0.12
13 Hot-smoked trout 73 Off-odour/off-avour LAB 6.8 0.30
14 Cooked and cold-smoked mackerel 75 Off-odour/off-avour LAB 7.4 +0.45
15 Cooked and hot-smoked cero (small mackerel) 150 Off-odour/off-avour Yeasts 4.9 0.03
16 Cooked marinated octopus with green olives 150 Off-odour/off-avour Yeasts 4.1 0.02
17 Cooked marinated octopus with pickled vegetables 150 Off-odour/off-avour 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.
llet the SSOs were yeasts, while in cold-smoked mackerel llet
withpepper the SSOs were lactic acidbacteria. The MSL was below
10
5.5
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 llet,
hot-smoked trout and cooked and cold-smoked mackerel, had
a shelf life rangingfrom73 to 81 days (Table 3). Lactic acidbacteria
were the SSOs with MSL above 10
6
CFU g
1
in all cases (Table 3).
These products were lightly preserved, allowing micro-organisms
to grow and reach levels that can cause microbial spoilage.
DISCUSSION
The differences found in moisture and NaCl contents, a
w
, pH,
smoking intensity, etc. of processed sh products denitely
affect their shelf life.
11
Shelf lives higher than 150 days have
been reported for products such as marinated sardines
12
and
salted anchovies,
13
while for other traditional products such as
salted bonito (lakerda) the shelf life was about 90 days.
14
Lightly
preserved products such as cold-smoked salmon had a shelf
life shorter than 50 days.
1517
The shelf life of cold-smoked sh
varies with species, degree of smoking and salt content.
16,17
Smoking and/or heat treatment did not affect the shelf life of our
products, at least not to the extent that pH and salt content did. It
seems that these processes, especially heating and hot smoking,
enhance the reduction in initial microbial load but do not affect
the growth of the remaining microbial populations, which are
inhibited by the intrinsic properties (pH and NaCl content) only.
Indeed, Kilinc and Cackli
18
found that pasteurisation did not
affect the shelf life of sardine marinades stored at 4

C, while
increased salt content extended the shelf life of cold-smoked
salmon.
17
Traditionally, processed seafoods are ready-to-eat products,
so microbiological safety is of great concern. Staphylococci
and enteric pathogens such as salmonellae might contaminate
the products as a result of poor hygiene conditions, cross-
contamination from personnel and other sources, etc. Salmonella
and Staphylococcus aureus can survive for long time in seafoods
with low a
w
.
19,20
Enterobacteriaceae and S. aureus can also
withstandlowpHfor quitealongtimeduringstorageof marinated
Pacic saury.
7
The very low populations of total coliforms and
S. aureus (belowdetectionlimit) foundinour products conrmthe
good hygiene conditions during manufacturing and imply that
the possibility of the presence of enteric pathogens is very low.
However, it is well established that pathogenic micro-organisms
can adapt and develop resistance to hurdles such as low a
w
and pH,
21
and ready-to-eat seafood products have also been
associated with listeriosis outbreaks.
22
To document the safety of
such products, a different experimental design is required, but this
was not among the aims of the present work.
The selection of spoilage microbiota was affected by low pH
and NaCl content. Spoilage of fresh sh is caused by metabolic
activity of micro-organisms such as Pseudomonas spp. and
Shewanella putrefaciens.
3
However, in our products the SSOs
were lactic acid bacteria and yeasts. It is widely accepted that
hurdles such as low pH and a
w
inhibit the spoilage Gram-
negative microbiota, while they favour the growth of lactic acid
bacteria and yeasts.
23
As the intensity of preservation hurdles
is increased compared with fresh sh, changes from Gram-
negative bacteria to lactic acid bacteria and yeasts have been
recorded.
24
Lactobacillus spp. were the predominant microbiota
in marinated anchovies, sardines and pacic saury,
7,18,25
and
Lactobacillus alimentarius was the SSO in marinated herring.
26
Lactic acid bacteria were the main spoilage microbiota of salted
anchovies,
27
cold-smoked trout
28
and salmon,
17,29
while yeasts
were found in salted bonito.
14
Other micro-organisms have
also been reported as part of the spoilage microbiota, such as
Enterobacteriaceae, staphylococci and halophilic pediococci in
salted anchovies.
27,30
Enterobacteriaceae and B. thermosphacta
together with Lactobacillus spp. and yeasts have been reported in
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cold-smoked salmon,
15,17,29
while Micrococcus spp. together with
Lactobacillus spp. have been found in marinated anchovies.
25
It can be seen from the present study that the population
level of dominant spoilage micro-organisms at the end of shelf
life was dependent on the intensity of the hurdles applied. In
products with lowpHor high NaCl content, microbial populations
hardly reach 10
6
CFU g
1
. The marination process and its
intensity greatly affect the survival and growth of microbial
populations. Populationsusuallydropbelowdetectionlimit during
marination,
31
while the spoilage population can range from 10
5
to 10
2
CFU g
1
.
7,18,32,33
As an exception, acid-resistant spoilage
populations as high as 10
7
and 10
9
CFU g
1
were reported
in marinated and acetic acid-preserved herring respectively.
26,34
Salts and brines also suppress the maximumpopulation density of
spoilage bacteria. In traditional salted anchovies, total mesophilic
and lactic acid bacteria reached populations of about 10
6
and
10
3.5
CFU g
1
respectively,
27
while in brined anchovies the
microbial population level was affected by brine concentration:
the higher the brine concentration, the lower the microbial
spoilage population.
13
In cold-smoked salmon, which is a lightly
processed seafood, populations of about 10
7
10
8
CFU g
1
have
been reported.
1517,29
Spoilage of fresh sh is caused by metabolic activity of
micro-organisms such as Pseudomonas spp., S. putrefaciens,
Photobacterium phosphoreum, etc., with an MSL usually above
10
7
CFU g
1
.
3,4
The lower MSL in most of our samples implies
that mechanisms other than microbial activity might contribute
to the quality deterioration. Despite microbial activity being the
main cause of spoilage of lightly preserved products such as cold-
smoked salmon, autolytic changes can also play a role.
35
Various
chemical reactions such as lipid oxidation may contribute to the
spoilage of processed sh 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 anchovies
13
and
hot-smoked tilapia.
36
The application of hurdles such as low
pH and a
w
, 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 smoked
mackerel was inhibited, in contrast to the TBA value.
37
Yanar
et al.
36
also found that oxidative rancidity increased as a result
of increasing salt content. It seems that mechanisms other
than microbial activity, such as autolytic changes and chemical
oxidation, might contribute to the quality deterioration of such
products.
CONCLUSIONS
Despite the fact that the shelf life of traditionally processed sh
products varies with moisture content, NaCl concentration, pH, a
w
and smoking and heating intensity, it seems that primarily the pH
andsecondarilytheNaCl content determinedtheshelf lifeduration
of our products. The spoilage microbiota was dominated by lactic
acid bacteria and yeasts. The higher the intensity of preservation
hurdles, the lower is the contribution of microbial activity to
spoilage mechanisms and shelf life duration. The contribution of
chemical oxidation and autolysis to spoilage and shelf life should
be studied in future work.
ACKNOWLEDGEMENT
We would like to thank NIREAS SA, Greece for the provision of
seafood products.
REFERENCES
1 Leistner L, Basic aspects of food preservation by hurdle technology.
Int J Food Microbiol 55:181186 (2000).
2 Ashie INA, Smith JP and Simpson BK, Spoilage and shelf life extension
of fresh sh and shellsh. Crit Rev Food Sci Nutr 36:87121 (1996).
3 Gram LandHuss HH, Microbiological spoilageof shandshproducts.
Int J Food Microbiol 33:121137 (1996).
4 Gram L and Dalgaard P, Fish spoilage bacteria problems and
solutions. Curr Opin Biotechnol 13:262266 (2002).
5 K ose S, Evaluation of seafood safety health hazards for traditional sh
products: preventive measures and monitoring issues. Turk J Fish
Aquat Sci 10:139160 (2010).
6 Gram L, Microbiological spoilage of sh and seafood products, in
Compendium of the Microbiological Spoilage of Food and Beverages,
ed. by Spenser WH and Doyle MP. Springer Science, New York, NY,
USA, pp. 87119 (2009).
7 Sallam KI, Effect of marination process on the microbiological quality
of Pacic saury (Cololabis saira) during vacuum-packaged storage
at 4

C. Int J Food Sci Technol 43:220228 (2008).

8 Huss HH, Assurance of seafood quality. FAO Fisheries Technical Paper
No. 34 (1994).
9 Gram L, Trolle G and Huss HH, Detection of specic spoilage bacteria
from sh stored at low (0

C) and high (20

C) temperatures. Int J
Food Microbiol 4:6572 (1987).
10 Dalgaard P, Gram L and Huss HH, Spoilage and shelf life of cod llets
packed in vacuum or modied atmospheres. Int J Food Microbiol
19:283294 (1993).
11 Fuentes A, Fern andez-Segovia I, Barat JM and Serra JA, Physicochem-
ical characterization of some smoked and marinated sh products.
J Food Process Preserv 34:83103 (2010).
12 G oko glu N, Cengiz E and Yerlikaya P, Determination of the shelf life of
marinated sardine (Sardina pilchardus) stored at 4

C. Food Control
15:14 (2004).
13 Karacam H, Kutlu S and K ose S, Effect of salt concentration and
temperature on the quality and shelf life of brined anchovies.
Int J Food Sci Technol 37:1928 (2002).
14 Erkan N, Tosun YS , Alakavuk

UD and Ulusoy S , Keeping quality of
different packaged salted Atlantic bonito lakerda. J Food Biochem
33:728744 (2009).
15 Leroi F, Joffraud JJ, Chevalier F and Cardinal M, Research of quality
indices for cold-smoked salmon using a stepwise multiple
regression of microbiological counts and physico-chemical
parameters. J Appl Microbiol 90:578587 (2001).
16 Leroi F and Joffraud JJ, Salt and smoke simultaneously affect chemical
and sensory quality of cold-smoked salmon during 5

C storage
predicted using factorial design. J Food Protect 63:12221227
(2000).
17 Hansen LT, Gill T and Huss HH, Effect of salt and storage temperature
on chemical, microbiological and sensory changes in cold-smoked
salmon. Food Res Int 28:123130 (1995).
18 Kilinc B and Cackli S, The determination of the shelf life of pasteurized
and non-pasteurized sardine (Sardina pilchardus) marinades stored
at 4

C. Int J Food Microbiol 40:265271 (2005).

19 Arkoudelos JS, Samaras FJ and Tassou CC, Survival of Staphylococcus
aureus and Salmonella Enteritidis on salted sardines (Sardina
pilchardus) during ripening. J Food Protect 66:14791481 (2003).
20 Mol S, Cosansu S, Alakavuk DU and Ozturan S, Survival of Salmonella
Enteritidis during salting and drying of horse mackerel (Trachurus
trachurus) llets. Int J Food Microbiol 139:3640 (2010).
21 Juneja VK and Novak JS, Adaptation of foodborne pathogens to stress
from exposure to physical intervention strategies, in Microbial
Adaptation to Stress and Safety of New-generation Foods, ed. by
Yousef AE andJuneja VK. CRCPress, Boca Raton, FL, USA, pp. 3154
(2003).
22 Timothe J, Nightingale KK, Gall K, Scott VNandWiedmann M, Tracking
of Listeria monocytogenes in smoked sh processing plants. J Food
Protect 67:328341 (2003).
23 Jay JM, Loessner MJ and Golden DA, Modern Food Microbiology (7th
edn). Springer Science+Business Media, New York, NY (2005).
24 Gram L, Microbiological food spoilage, in Handbook of Food Science,
Technology and Engineering, ed. by Hui YH. Taylor and Francis/CRC
Press, Boca Raton, FL, USA, pp. 51-151-16 (2005).
25 Fuselli SR, Casales MR, Fritz R and Yeannes MI, Isolation and
characterization of microorganisms associated with marinated
anchovy (Engraulis anchoita). J Aquat Food Prod Technol 7:2938
(1998).
J Sci Food Agric 2013; 93: 11841190 c 2012 Society of Chemical Industry wileyonlinelibrary.com/jsfa
1
1
9
0
www.soci.org IS Boziaris, AK Stamatiou, GJE Nychas
26 Lyhs U, Korkeala H, Vandamme P and Bjorkroth J, Lactobacillus
alimentarius: a specic spoilage organism in marinated herring.
Int J Food Microbiol 64:355360 (2001).
27 Aponte M, Blaiotta G, Francesca N and Moschetti G, Could halophilic
archaea improve the traditional salted anchovies (Engraulis
encrasicholus L.) safety and quality? Lett Appl Microbiol 51:697703
(2010).
28 Lyhs U, Bj orkroth J and Korkeala H, Characterization of lactic acid
bacteria from spoiled vacuum-packaged, cold-smoked rainbow
trout using ribotyping. Int J Food Microbiol 52:7784 (1999).
29 Hansen LT and Huss HH, Comparison of the microora isolated from
spoiled cold-smoked salmon fromthree smokehouses. Food Res Int
31:703711 (1998).
30 Villar M, de Ruiz Holgado AP, Sanchez JJ, Trucco RE and Oliver G,
Isolation and characterization of Pediococcus halophilus fromsalted
anchovies (Engraulis anchoita). Appl Environ Microbiol 49:664666
(1985).
31 Fuselli SR, Casales MR, Fritz R and Yeannes MI, Microbiology of
the marination process used in anchovy (Engraulis anchoita)
production. LebensmWiss Technol 27:214218 (1994).
32 Poligne I and Colligan A, Quick marination of anchovies (Engraulis
enchrasicolus) using acetic and gluconic acids. Quality and stability
of the end products. LebensmWiss Technol 33:202209 (2000).
33 Cadun A, Cakli S and Kisla D, A study of marination of deepwater pink
shrimp(Parapenaeus longirostris, Lucas, 1846). FoodChem90:5359
(2005).
34 Lyhs U, Koort JMK, Lundstr om HS and Bj orkroth JK, Leuconostoc
gelidum and Leuconostoc gasicomitatum strains dominated the
lactic bacteriumpopulation associated with strong slime formation
in an acetic-acid herring preserve. Int J Food Microbiol 90:207218
(2004).
35 Hansen LT, Gill T, Rntved SD and Huss HH, Importance of autolysis
and microbiological activity on quality of cold-smoked salmon.
Food Res Int 29:181188 (1996).
36 Yanar Y, C elik M and Akmca E, Effect of brine concentration on shelf
life of hot-smokedtilapia (Oreochromis niloticus) storedat 4

C. Food
Chem97:244247 (2006).
37 Goulas AE and Kontominas MG, Effect of salting and smoking-method
on the keeping quality of chub mackerel (Scomber japonicus):
biochemical and sensory attributes. FoodChem93:511520 (2005).
wileyonlinelibrary.com/jsfa c 2012 Society of Chemical Industry J Sci Food Agric 2013; 93: 11841190