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FRESHNESS QUALITY AND SPOILAGE OF CHILL-STORED FISH

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FRESHNESS QUALITY AND SPOILAGE OF CHILL-STORED FISH

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Finfish are generally regarded as being much more perishable than other high-protein muscle foods. Wet fish, whether marketed in the whole, uneviscerated condition or in some dressed or split form, will undergo rapid quality deterioration from harvesting to retailing, particularly if not properly handled and stored. The spoilage of chilled fish is a complex process resulting from the composite activities of autolysis, spontaneous chemical reactions, bacterial attack and leaching by ice-melt water. These effects proceed concurrently and independently, their relative importance varying with species of fish and its environment, intrinsic composition, storage conditions, harvesting method and handling procedures. An understanding of the mechanisms involved in spoilage is essential in order to formulate effective strategies for regulating quality and controlling adverse changes to it. Freshness, defined in terms of odor, flavor, texture and appearance, makes a major contribution to the overall quality of fish. Placing on the market fresh fish products, the sensory attributes of which have changed as little as possible from those generally regarded as contributing to peak quality post-harvest, requires that different freshness levels of the raw material can be determined consistently and accurately. This is particularly important when fish at some intermediate stage of deterioration have to be graded for processing and distribution. The industry has long sought a reliable method for assessing freshness specification of the starting material and making sure that the product will not become stale when distributed and displayed. An ideal method should be rapid and accurate, cheap, non-destructive, and preferably applicable to all seafood; it should correlate freshness quality with the time and temperature of storage following the harvest and should provide a basis for estimating future storage life. However, this is very difficult to achieve in view of the variety of commercial fish species and the complex of interrelated sensory, chemical and microbiological changes determining the stage of spoilage.
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In: Food Policy, Control and Research ISBN 1-59454-408-5
Editor: Arthur P. Riley, pp. 35-86 © 2005 Nova Science Publishers, Inc.
Chapter 2
FRESHNESS QUALITY AND
SPOILAGE OF CHILL-STORED FISH
V.P. Lougovois and V.R. Kyrana
Fisheries Laboratory
Department of Food Technology, T.E.I. of Athens
Agiou Spyridonos, 122 10 Egaleo
Athens, Greece
ABSTRACT
Finfish are generally regarded as being much more perishable than other high-protein
muscle foods. Wet fish, whether marketed in the whole, uneviscerated condition or in
some dressed or split form, will undergo rapid quality deterioration from harvesting to
retailing, particularly if not properly handled and stored. The spoilage of chilled fish is a
complex process resulting from the composite activities of autolysis, spontaneous
chemical reactions, bacterial attack and leaching by ice-melt water. These effects
proceed concurrently and independently, their relative importance varying with species
of fish and its environment, intrinsic composition, storage conditions, harvesting method
and handling procedures. An understanding of the mechanisms involved in spoilage is
essential in order to formulate effective strategies for regulating quality and controlling
adverse changes to it.
Freshness, defined in terms of odor, flavor, texture and appearance, makes a major
contribution to the overall quality of fish. Placing on the market fresh fish products, the
sensory attributes of which have changed as little as possible from those generally
regarded as contributing to peak quality post-harvest, requires that different freshness
levels of the raw material can be determined consistently and accurately. This is
particularly important when fish at some intermediate stage of deterioration have to be
graded for processing and distribution. The industry has long sought a reliable method
for assessing freshness specification of the starting material and making sure that the
product will not become stale when distributed and displayed. An ideal method should
be rapid and accurate, cheap, non-destructive, and preferably applicable to all seafood; it
should correlate freshness quality with the time and temperature of storage following the
harvest and should provide a basis for estimating future storage life. However, this is
V.P. Lougovois and V.R. Kyrana
very difficult to achieve in view of the variety of commercial fish species and the
complex of interrelated sensory, chemical and microbiological changes determining the
stage of spoilage.
INTRODUCTION
While safety and wholesomeness are indisputably the most important parameters of
quality, sensory characteristics such as odor, flavor and palatability are the major attributes
consumers can readily pass judgment on in their acceptance or rejection of the fishery
product. The deterioration in sensory quality of chill-stored wet fish results from natural
processes which are quite independent of man’s deliberate intervention and are perceived as
loss of freshness and development of spoilage. Post-mortem, the immediate changes are
chemical and enzymic autolytic processes. Autolysis brings about various structural
alterations in the tissues, including rigor mortis and different degrees of disintegration of the
muscle ultra structure. Autolytic changes in carbohydrates, nucleotides, lipids and lipid-
derived compounds reduce the desirable odors and flavors associated with freshly harvested
fish. Digestive enzymes from heavily feeding fish can penetrate the belly wall causing
softening of the adjacent musculature, unsatisfactory flavors and discoloration, whilst in
certain physiological conditions softening may be due to endogenous enzymes. On the other
hand, fatty-type fish may undergo lipid oxidation rapidly at refrigerator temperatures, due to
the highly unsaturated nature of the fatty acids in fish tissue.
The autolytic and chemical reactions certainly cause quality deterioration but are not
responsible for the offensive fishy, rotten, sulfide odors and slimy and pulpy texture of
spoiled fish. These spoilage phenomena, limiting storage life of wet fish, result from
bacterial activity, in particular reduction of trimethylamine oxide to trimethylamine,
oxidative deamination of amino acids and peptides to ammonia, release of fatty acids,
degradation of proteins and breakdown of sulfur-containing amino acids to methyl
mercaptan, dimethylsulfide and hydrogen sulfide. Fish tissues contain high proportions of
free amino acids and other non-protein nitrogenous constituents, which are utilized actively
by bacteria during spoilage. In addition, fish muscle is low in less digestible connective
tissue and has a high water activity and near neutral pH, thus providing an excellent substrate
for the growth of most heterotrophic bacteria. The composition of free amino acids in fish
flesh can influence the pattern of spoilage and can impact human health through the
formation of biogenic amines.
This chapter discusses in detail the mechanisms (enzymic-autolytic, chemical and
microbial) involved in the deterioration of chill-stored fish, and the changes in sensory
attributes accompanying spoilage. Factors affecting keeping quality and potential storage
life, including species, physical stress during capture, primary processing operations and
storage temperature, are also discussed. The chapter reviews methods (sensory,
chemical/biochemical, physical and microbiological) that may be used to assess freshness
quality and storage stability of wet finfish. Potential public health concerns associated with
fresh fish, e.g. pathogenic bacteria, ciguatera and algal intoxications and parasites that can
infect humans, are not discussed here.
2
Freshness Quality and Spoilage of Chill-Stored Fish
AUTOLYSIS OF FISH MUSCLE
Although bacterial metabolism is largely responsible for rendering the fish inedible, it is
the highly active endogenous enzymes in the tissues, blood, viscera and skin that provide
substrates necessary to sustain bacterial growth. Many of the decomposition reactions can be
catalyzed both by endogenous and bacterial enzymes, and thus it is not always possible to
distinguish precisely between autolysis and bacterial changes.
It is generally the case that in marketed fish adenosine triphosphate (ATP) has long since
disappeared and, as a consequence, the ATP-dependent processes that characterize the living
muscle cell have ceased to take place. Many features of the muscle that are important from
the consumer’s point of view are determined by the manner in which this ATP-depleted state
is attained. Accordingly, the metabolic processes occurring as ATP-replete muscle is
transformed into ATP-free flesh have an important bearing on the food quality of the muscle.
Post-Mortem Metabolism and the Onset of Rigor Mortis
Upon the death of the animal the heart stops beating and as a result the circulatory
system ceases to supply the muscles with oxygen and metabolizable fuels, such as glucose.
Since no oxygen is available for normal respiration the mitochondrial system ceases to
function in all but surface cells [1]. The production of energy from ingested nutrients is
greatly restricted and this rapidly brings about important changes in the muscle tissue. In
order to replenish the ATP which is being continually hydrolyzed to power the various
energy-consuming activities that constitute cellular work, skeletal muscle cells have
subsequently to rely upon the anaerobic metabolism of their intracellular fuel [2]. For most
teleost fish, glycolysis is the only possible pathway for the production of energy once the
circulatory system has been disrupted. One additional source of ATP re-synthesis is the
reservoir of creatine phosphate (CP). The conversion of CP to creatine and the transfer of its
phosphate to adenosine diphosphate (ADP) temporarily regenerate some ATP. Once the
creatine phosphate has been used up, which occurs fairly rapidly, anaerobic glycolysis
continues to regenerate some ATP with the end product, lactate, accumulating.
The breakdown of glycogen to glucose-1-phosphate (glycogenolysis) and its subsequent
conversion via the glycolytic pathway to pyruvate and NADH is accompanied by the re-
synthesis of a small quantity of ATP (2 or 3 moles of ATP per mole of glucose) in so-called
substrate-level phosphorylation [3]. This sequence of events is essentially the same as in
vivo, when the supply of oxygen may become temporarily impaired in the exerted muscle.
Glycolysis may cease because of depletion of substrates or because certain glycolytic
enzymes cease to function due to the drop in pH caused by the hydrolysis of ATP. As
glycolytic activity slows down, ATP concentration decreases, mainly due to the continuing
action of various ATPases in the membrane systems, with most of the nucleotide depleted in
24 hours or less. As a result of the post-mortem depletion of ATP, biosynthetic reactions
come to a halt, and there is a loss of the cell’s ability to maintain its integrity, especially with
respect to membrane systems.
Both ATP and ADP act as plasticizers for actin and myosin, thus preventing their
interaction and keeping the muscle in a state of relaxation. When the intracellular level of
3
V.P. Lougovois and V.R. Kyrana
these nucleotides declines post-mortem below 1.0 μmole per g tissue, actin and myosin
interact and the muscle enters rigor mortis [4]. The muscle, which just after death is pliable,
limp and elastic, turns stiff, hard and inextensible, and cannot be stretched significantly
without breaking. The body of the fish in rigor often has a bent form due to the tension
developed by antagonistic muscles, making machine handling impossible. During rigor the
filleting yield will be very poor, and rough handling will increase the incidence of
mechanical gaping. Some myocommata, connecting the myotomes of the muscles, break
under the contraction associated with rigor mortis and as a result the flakes come apart. If the
fillets are excised pre-rigor, the muscles can contract freely and the fillets will shrink
following the onset of rigor. Length shortening of mackerel and hake fillets, excised just
after hauling on board, is 6-17%, while in some deep-water species up to 40% shrinkage may
be encountered, depending on the duration of the trawling and the load of fish in the net [5];
pre-rigor filleted salmon fillets have been found to shrink by approximately 15% of the
original length [6].
Although the hydrogen ions generated in post-mortem muscle come from the hydrolysis
of ATP and not from the production of lactate, there is a strong relationship between the drop
in pH and lactate concentration in the muscle [1]. This is because the amount of lactate
formed is roughly proportional to the amount of ATP produced by the glycolytic system. On
the other hand, the amount of lactic acid produced is related to the amount of stored
carbohydrate (glycogen) in the living tissue [7]. Pre-capture stress has been reported to
accelerate pH decline in the muscle post-mortem [8]. However, the final pH attained,
referred to as “ultimate pH”, depends on the type of muscle and fish species but (unlike
mammalian muscle) is unrelated to the degree of stress or exercise prior to death [9]. Lactate
produced as a result of struggling during harvest is only very slowly removed from the
muscle. According to Hultin [1], when a fish struggles extensively before death, much
lactate can be produced and this lactate, for the most part, is present in the muscle at the time
of death. On the other hand, a fish that struggled only slightly prior to harvest would contain
only a small amount of lactate at the point of death, but normal post-mortem glycolysis
would cause muscle lactate to increase to essentially the same level as that existing in fish
that struggled vigorously prior to death.
In lean white fish (e.g. cod) the ultimate pH of the muscle is normally 6.2-6.6, while in
red meat species (e.g. mackerels and tuna) this value tends to be low (5.5-5.9). In general,
fish muscle contains relatively low levels of glycogen compared to mammals, primarily due
to changing feeding patterns with season. Thus, the ultimate pH is normally higher in fish
than in warm-blooded animals. The post-mortem lowering of pH bears an important
influence on various quality aspects of the muscle, including texture, water binding capacity
and resistance to microbial growth.
The time post-mortem for rigor onset (incipient loss of extensibility) and resolution is
affected by such factors as species, animal to animal variation within a species, type of
muscle, temperature, handling, size, pre-slaughter stress and biological status of the fish [10-
13]. In well nourished, not exhausted, large, refrigerated fish, rigor develops later, is more
intensive and lasts longer than in small fish of poor physical condition kept at ambient
temperature. When the fish is starved and the glycogen reserves are depleted, rigor mortis
starts shortly after death. Stiffness generally begins in the head region of the fish and
gradually spreads towards the caudal muscles, even though the reverse sequence has also
been observed [5]. Killing the fish by immersing in ice-cold water (hypothermia) gives the
4
Freshness Quality and Spoilage of Chill-Stored Fish
fastest onset of rigor, whereas a blow on the head may give a delay of up to 18 hours [14].
Destruction of the brain of fish that have not been subject to prolonged struggle delays
significantly the onset of rigor.
The effect of temperature on rigor is not uniform [15]. In cold-water species, high
storage temperatures give a fast onset and a very strong rigor mortis, often causing
weakening of the connective tissue and gaping of the fillet. For cod, the critical temperature
above which gaping increases sharply is 17 °C. On the other hand, observations on tropical
fish have shown that the onset of rigor is accelerated at 0 °C compared to 10 °C [16, 17]. It
has been demonstrated that the lower the difference between acclimation and storage
temperatures, the longer the period needed to reach maximal rigor and rigor strength at
storage temperatures below 10 °C [18].
When muscle is allowed to stand post-rigor at refrigerated temperatures, it gradually
softens and becomes pliable again, but no longer as elastic as before rigor. This tenderization
process, referred to as “resolution of rigor, is extremely significant in the marketing of meat
since it directly influences the texture of the cooked muscle. Understanding the mechanisms
involved in the delay, onset, and resolution of rigor mortis could help to improve the textural
quality of fish.
Degradation of Nucleotide Metabolites
The main route of ATP degradation in finfish muscles post-mortem involves gradual and
stepwise dephosphorylation to adenosine mono-phosphate (AMP) and deamination to
inosine mono-phosphate (IMP). The latter degrades to inosine (Ino) and eventually to
hypoxanthine (Hx), which may be degraded further to xanthine and uric acid [20]. Although
degradation proceeds in the same manner with most finfish, the rate of the individual
reactions varies greatly from one species to another. Nucleotide metabolism commences
upon death and its rate is influenced by factors such as the amount of struggling prior to
death, not bleeding the fish and temperature at which the fish is stored [8]. Fish handling
may accelerate the breakdown of nucleotide metabolites by rendering substrates more
accessible to enzyme action.
The hydrolysis of ATP to IMP (through AMP) is catalyzed entirely by endogenous
tissue enzymes and in chilled fish is nearly complete within 24 hours after harvesting [20].
Further stages are rather slow, although in the breakdown of IMP and inosine, besides tissue
enzymes, bacteria are also involved. Spoilage strains of Pseudomonas and Shewanella have
been shown to decompose IMP, inosine and hypoxanthine, thus casting some doubt on the
explicitly endogenous basis of nucleotide degradation [21, 22]. Proteus vulgaris are also
capable of producing nucleoside phosphorylase and thus may accelerate the conversion of
inosine to hypoxanthine, particularly during the later stages of storage [23].
However, a substantial amount of microbial enzymes is not a prerequisite for nucleotide
metabolism [24]. The exclusion of bacteria from fresh fish might inhibit gross spoilage but it
would not prevent the product from rapidly becoming unacceptable, as autolytic enzymes
seem to play a major role in the loss of desirable odors and flavors in fresh fish [25-27].
5
V.P. Lougovois and V.R. Kyrana
HARVEST OF FISH
DEATH
BLOOD CIRCULATION CEASES
OXYGEN SUPPLY FAILS
OXIDATION-REDUCTION POTENTIAL FALLS
AEROBIC RESPIRATION CEASES GLYCOGEN → LACTIC ACID
ATP LEVEL DIMINISHES pH FALLS
ONSET OF
RIGOR MORTIS
CATHEPSINS LIBERATED
AND ACTIVATED
ACCUMULATION OF HYPOXANTHINE PROTEINS → AMINO ACIDS
BACTERIAL GROWTH
ACCUMULATION OF METABOLITES
Figure 1. Consequences of circulatory failure in fish muscle tissue (adapted from [19])
Changes in Proteins and other Nitrogenous Constituents
The major proteolytic enzymes in fish muscle, the cathepsins, are acid proteases found
mostly in the lysozomes. The optimal pH of activity of these enzymes is much lower than the
pH normally encountered in fish and therefore, their autolytic activity in post-mortem muscle
is rather low.
However, cathepsins D and L are believed to play a major role in the autolytic
degradation of fish tissue, since they are active over a wide pH range. Cathepsin D is active
over the pH range 3-8 with a maximum near pH 4.0 [28, 29]. The enzyme can initiate the
degradation of myofibrillar proteins to peptides, which may then be degraded further by
other enzymes. Cathepsin L has been implicated in the softening of salmon muscle during
spawning migration [30]; it has also been associated with the jellied meat condition
developing in flounder [31] and the softening of Pacific hake muscle infected by
myxosporidian parasites [32]. This enzyme contributes more to the autolysis of fish muscle
than cathepsin D, since it is far more active at neutral pH, and has been shown to digest both
myofibrillar proteins and connective tissue [4].
In small uneviscerated fish, autolytic changes are brought about not only by endogenous
muscle enzymes but also by kidney and liver cathepsins, as well as by digestive enzymes.
Advanced proteolytic breakdown caused by enzymes released from the alimentary canal,
6
Freshness Quality and Spoilage of Chill-Stored Fish
may lead to belly bursting in heavily feeding herring, sardine, anchovy and mackerel [33].
Several studies have demonstrated that the enzymes responsible for the rapid autolytic
degradation of the abdominal tissue are digestive proteases, especially acidic proteases from
the stomach and alkaline proteases from the pyloric ceca, pancreas and intestine [34-38]. The
phenomenon is accelerated by weakening of the connective tissue at the lower post-mortem
pH values encountered in fish caught during periods of heavy feeding [7].
Trimethylamine oxide (TMAO), found in almost all marine and some fresh water fish
species [39, 40], is typically reduced to trimethylamine (TMA) by spoilage bacteria.
Trimethylamine is an exceptionally potent aroma compound that has been long associated
with deteriorated flavor quality of marine fish [41]. In some species a trimethylamine N-
oxide aldolase (TMAOase) is present in the muscle tissue, which can break down TMAO to
dimethylamine (DMA) and formaldehyde [42-45]. The TMAOases are multi enzymic
systems commonly found in gadoid and related species and thus, the reaction proceeds faster
in the muscle tissue of cod, haddock, and hakes [46]. Most of the TMAOases appear to be
membrane-bound and become most active when the tissue membranes are disrupted by
mincing or freezing. Certain tissues, such as kidney, spleen and gall bladder, are extremely
rich in these enzymes [47-49].
In the muscles of sharks and rays, even in fresh fish, ammonia may accumulate due to
the activity of endogenous urease. Large amounts of ammonia are also generated in fish
muscles upon autolytic deamination of AMP.
MICROBIAL SPOILAGE
The Microbial Flora of Freshly Caught Fish
It is generally accepted that the muscle tissue of healthy, live fish is sterile [50], although
a few reports to the contrary exist [51]. The natural bacterial flora resides mainly in the outer
slime layer of the skin, on the gills, and in the gastro-intestinal tract of feeding fish. The total
microbial load is subject to seasonal variation and depends on the pollution and temperature
of the environment, on the method of catching and on the conditions of handling on board
fishing vessel [5].
The distribution of microorganisms in the sea and other large water masses is not
uniform. The water in the open sea normally contains very small numbers of bacteria, i.e. a
few colony forming units (CFU) cm
-3
, while the coastal regions and the sediments may be
heavily polluted, up to about 10
6
-10
7
CFU cm
-3
[52]. Increased bacterial populations in late
spring and autumn may be associated with plankton blooms, but high productivity could also
result in reduced numbers due to the antagonistic or antibiotic effects exerted by certain
planktonic forms on bacteria [53].
Bacterial numbers on the skin of fish captured individually in clean, cold surface waters
have been reported as one to ten CFU cm
-2
, whereas the total surface count on fish from
commercial bottom catches may reach numbers as high as 10
5
-10
6
CFU cm
-2
[5]. Trawled
fish usually carry microbial loads that are 10-100 times greater than those on line-caught fish
[54]. The bacterial load of the gills ranges from 10
3
to 10
5
CFU g
-1
and that of the intestines
from very few in non-feeding fish to 10
7
CFU g
-1
or greater in actively feeding species [55].
7
V.P. Lougovois and V.R. Kyrana
These values are quoted from plates incubated at 20-25 °C. With temperate or cold-water
fish, total numbers of viable bacteria decrease at incubation temperatures above 30 °C and at
37 °C the viable counts are about one tenth of the 20-25 °C count. Incubation temperatures
below 20 °C seem to make little difference, somewhat higher counts sometimes being
obtained at 0-5 °C [50]. The use of sea water or fresh water also makes little difference in the
count if sufficient salt (usually 0.5% NaCl) is included in the media. Fish from warm waters
frequently carry greater numbers of bacteria than cold-water fish and yield higher counts at
35-37 °C.
The population of microorganisms associated with living fish reflects the microflora of
the environment and of the fish’s feed at the time of capture or harvest, but is modified by
the ability of different microorganisms (mainly bacteria) to multiply in the sub-environments
provided by the skin surfaces, gill areas and the alimentary canal. Typically, bacteria from
skin and gills are predominantly aerobic, although facultative anaerobic bacteria, particularly
Vibrio spp., may occur in high numbers on pelagic fish [56]. Strictly anaerobic bacteria are
uncommon on the skin and gills of freshly caught fish but occur in significant numbers in the
intestine [22].
The poikilotherm nature of fish allows bacteria with a broad temperature range to grow.
The microbial flora on marine fish caught in temperate and northern hemisphere waters,
where the temperature of the seabed is 10 °C or less, is dominated by psychrotrophic Gram-
negative rod-shaped bacteria belonging to the genera Pseudomonas, Shewanella (previous
Alteromonas), Psychrobacter (Moraxella), Acinetobacter, Vibrio, Flavobacterium and
Cytophaga. Gram-positive organisms e.g. Bacillus, Micrococcus, Clostridium, Lactobacillus
and Corynebacterium can also be found in varying proportions [22, 57]. Fish from warm
(tropical and subtropical) waters often carry a major population of mesophilic Gram-positive
bacteria, particularly Bacillus, Micrococcus and Corynebacterium [58]. The Gram-negative
bacteria on warm-water finfish are similar to those on cold water fish [59-61].
The fish alimentary tract normally contains a much higher proportion of Vibrio strains,
often dominating the intestinal flora of marine fish, organisms formerly identified as
Achromobacter, Pseudomonas, Xanthomonas and Gram-positive microflora, mainly
Clostridium spp. and other spore-forming bacteria. Those reported include Clostridium
sporogenes, Cl. perfringens, Cl. bifermentans, Cl. tetani, Cl. tertium, Cl. lentoputrescens and
Cl. botulinum [5, 52, 53]. Members of the Enterobacteriaceae commonly found in the
intestine of warm-blooded animals are not normally isolated from marine finfish captured
away from coastlines [22].
The microflora of marine fish are predominantly halotolerant, rather than halophilic;
they can grow over a wide range of salt levels, but displaying optimal growth in the presence
of 2-3% sodium chloride. Chilling fish in melting ice alters the saline conditions in the
environment of the bacteria, favoring the persistence and growth of halotolerant species that
can adapt to changes in salt level [60].
The microbial flora of fresh-water fish can be quite variable, reflecting the microflora of
the water in which the animals live. The dominant organisms are those listed above for
marine finfish, except that Aeromonas replaces Vibrio. Fresh-water fish commonly carry
higher numbers of Streptococcus, Bacillus, Micrococcus and coryneforms than sea water fish
and, particularly if caught in warm waters, may also harbor members of the
Enterobacteriaceae [62]. The intestinal flora of fresh-water fish is dominated by Aeromonas
spp. In anadromus fish (e.g. salmon), which spend their adult life in the ocean but migrate in
8
Freshness Quality and Spoilage of Chill-Stored Fish
fresh water to spawn, Aeromonas and halotolerant Vibrio species alternate, reflecting the
change in water salinity [63].
In addition to bacteria, several genera of yeasts, mainly Rhodotorula, Torulopsis and
Candida, may also be present in small numbers among the surface microflora of fish. Yeasts
are fairly widespread both in fresh and salt waters, but literature on their occurrence on
living fish is very limited [64-67]. Fungi, except for some planktonic forms, appear restricted
mainly to estuarine and fresh waters and are rarely associated with fresh healthy marine fish.
Fungus infections are more common with diseased, dying freshwater fish, e.g. spawned out
Pacific salmon [50].
Growth of Spoilage Flora
Handling and refrigerated storage of wet finfish brings about a gradual change in the
number, distribution and composition of the microbial flora. Chilling exerts a selective
pressure on the bacterial populations on fish; at the temperature of melting ice (0°C),
mesophilic organisms grow very slowly, if at all, while the psychrotrophic strains increase
according to their capability to compete. The growth of bacteria follows a typical population
curve. After an initial lag period, the length of which depends primarily on storage
temperature and whether the fish were taken from cold or warm waters, the bacteria enter
exponential growth. Under aerobic storage conditions, the total count reaches numbers of
10
8
-10
9
CFU g
-1
flesh or cm
-2
of skin when spoilage is apparent [57].
The increase in bacterial population results to a large extent from a rapid growth of
Pseudomonas and Shewanella strains. As bacterial growth leading to spoilage progresses,
these Gram-negative psychrotrophic non-fermentative motile rods assume a dominant
position in the microflora since they are well adapted to refrigeration temperatures and
effectively utilize the extractives of fish flesh. During the later period of chill storage, 80-
90% of the population may consist of Pseudomonas and Shewanella strains; Psychrobacter
(Moraxella), Acinetobacter and Flavobacterium may also be present in smaller proportions.
Selection of this flora occurs almost irrespective of the initial composition, the main reason
being the low storage temperature [68]. The high iron-binding capacity of the siderophores
produced by pseudomonads may also cause this bacterial group to be positively selected in
an iron-limited environment such as fish muscle [69].
During harvesting and subsequent sorting, packing and storing operations, fish
microflora may show transient high relative proportions of Gram-positive bacteria, including
coryneforms, Micrococcus, Bacillus and Staphylococcus, as well as low levels of
Enterobacteriaceae. In the case of cold-water fish, both groups of bacteria derive from
environmental sources on the vessel (hooks, nets, deckboards, people etc.), but may be
naturally present on warm-water fish. The Gram-positive flora does not compete well with
psychrotrophs at chill temperatures, so that during later processing and refrigerated storage
the bacterial populations become again predominantly Gram-negative [22].
In the early days of storage, the bacterial population is confined almost entirely to the
outer and inner surfaces. There is no substantial evidence that invasion of tissues or blood
vessels occurs in whole or eviscerated fish held at low temperature [58]. Apparently, very
few bacteria invade the flesh and only at a rather late stage [70], the rate of penetration being
dependent largely on the barrier properties of the skin. An exception to this is when highly
9
V.P. Lougovois and V.R. Kyrana
active enzymes in the viscera of feeding fish digest the gut wall soon after death, permitting
entry of bacteria into the flesh surrounding the belly [71]. Slow penetration into the tissues of
intact fish may occur through skin cuts, puncture wounds and abrasions resulting from rough
handling or the death struggle. The exposed muscle layers of dressed or split fish and fish
fillets are especially vulnerable to invasion but most activity still occurs at the surfaces,
reflecting the oxidative nature of many bacterial processes leading to quality deterioration.
Under chill storage conditions, microbial spoilage is mainly a surface phenomenon resulting
to a large extent from the bacterial enzymes diffusing into the flesh and nutrients diffusing to
the outside [62].
Spoilage bacteria apparently have little difficulty in growing in the slime and on the
outer integument of fish. Slime is composed of mucopolysaccharide components, free amino
acids, trimethylamine oxide, piperidine derivatives and other related compounds [51].
Bacteria on fish surfaces metabolize surrounding nutrients, producing the volatile
compounds associated with spoilage. Thus, in the early days of storage, not the bacteria but
the products of their metabolism penetrate into the deeper layers of the flesh [5]. However,
under temperature abuse conditions, bacteria significantly invade the relatively sterile muscle
tissue through the lateral line pores, the collagen fibers as well as the gut, resulting in more
rapid spoilage [72, 73].
Specific Spoilage Organisms
Comparison of the chemical compounds developing in naturally spoiling fish and sterile
fish have shown that most of the volatile compounds responsible for the objectionable odors
perceived as spoilage are produced by bacterial metabolism [74]. However, not all strains of
bacteria occurring in spoiling fish are capable of producing objectionable odor changes
detected by sensory methods [68, 75]. The bacteria most commonly identified with spoilage
of iced raw finfish are species of Shewanella and Pseudomonas, with Shewanella
putrefaciens predominating at lower storage temperatures [22, 76-78]. These organisms are
most active, they are numerically predominant in the microflora of spoiling fish and their
ability to produce typical spoilage odors has been demonstrated by inoculation of individual
strains on to sterile fish muscle blocks or in muscle press juice at low temperatures [79].
Some Pseudomonas strains produce sulfide or ammonia odors, some produce fruity odors
due to esters of short chain fatty acids, and Shewanella strains produce strong ammonia and
putrid odors. On the other hand, Psychrobacter (Moraxella) and Acinetobacter strains
produce no significant odor changes though they grow well [53, 68]. Thus, regardless of the
differences in the initial microflora, the spoilage patterns of finfish stored aerobically in ice
are usually quite similar and are caused by Pseudomonas spp. and Shewanella putrefaciens
[57].
At elevated temperatures (10-37 °C) the microflora of spoiling fish is dominated by
fermentative mesophilic Vibrionaceae, and particularly if the fish are caught in polluted
waters, by mesophilic Enterobacteriaceae [78, 80, 81]. Shewanella putrefaciens may also
take part in the spoilage of ambient-stored marine fish [82]. Studies on the ambient spoilage
of freshwater fish have identified Aeromonas hydrophila as specific spoilers of rainbow trout
held at 37 °C [83]. Motile aeromonads are the specific spoilage bacteria of ambient-stored
Lake Victorian Nile perch [61].
10
Freshness Quality and Spoilage of Chill-Stored Fish
In environments with less oxygen available, the number of Pseudomonas spp. is reduced
but facultative anaerobes such as Shewanella putrefaciens and Photobacteriun phosphoreum,
which are capable of using trimethylamine oxide as the terminal electron acceptor in an
anaerobic respiration, grow to levels of 10
6
-10
8
CFU g
-1
[84, 85]. Vacuum-packed fish from
temperate marine waters, stored at low temperatures, are normally spoiled by these two
bacteria and differences in initial counts probably decide which of the two becomes most
important [57]. P. phosphoreum is a luminous, heat sensitive marine vibrio (salt-requiring),
which produces 30 times more trimethylamine per cell than S. putrefaciens [86]. It is easily
isolated from the intestines of various fish but its development varies considerably among
species and fish in the same catch [60]. An increased development of trimethylamine is
commonly seen under vacuum-packaging conditions, while the storage life is unaffected
compared to aerobically stored fish [85].
In CO
2
-enriched environments, the development of respiratory organisms like
Pseudomonas spp. and S. putrefaciens is greatly inhibited and their numbers rarely exceed
10
5
-10
6
CFU g
-1
. Nonetheless, only limited extension of storage life is achieved by packing
marine fish from temperate waters in a CO
2
-modified atmosphere, due to the presence and
subsequent growth of CO
2
-resistant P. phosphoreum to levels of 10
7
-10
8
CFU g
-1
[87, 88].
Gram-positive organisms, mainly lactic acid bacteria and Brochothrix thermosphacta,
are likely spoilers of vacuum-packed and CO
2
-packed fish from fresh or warmer waters,
whereas P. phosphoreum appears to be less important [89-94]. However, as trimethylamine
can be detected during the later stages of storage, it has been suggested that TMAO-reducing
organisms must be present at some level [57].
A number of mathematical models describing the growth of Pseudomonas spp., S.
putrefaciens, P. phosphoreum and Brochothrix thermosphacta have been developed for
predicting the time to rejection or remaining storage life of aerobically-stored and CO
2
-
packed fresh finfish [95-99].
Chemistry of Fish Spoilage
The primary substrate for bacterial growth and the main source of spoilage products is
the extractive fraction of the fish muscle. The group comprises primarily sugars and low
molecular weight nitrogenous constituents such as free amino acids and nucleotides,
trimethylamine oxide, creatine, taurine, the betaines, uric acid, anserine, carnosine and, in the
case of cartilaginous fish, urea. These compounds are more vulnerable to the action of
bacteria than proteins, so that their content and nature can influence the spoilage pattern of
stored fish [51]. Examples of substances produced as a result of bacterial metabolism of
muscle extractives are trimethylamine from trimethylamine oxide; volatile sulfides from
sulfur-containing amino acids; lower fatty acids from sugars (glucose, ribose) and lactate;
various carbonyl compounds from lipids and amino acids; hypoxanthine from inosine or
inosine monophosphate and various amines and ammonia from amino acids [22, 53, 78].
Trimethylamine reacts with lipids in the muscle, and along with volatile fatty acids,
contributes to the characteristic fishy odor of stale fish. Aeromonas spp., psychrotolerant
Enterobacteriaceae, P. phosphoreum, S. putrefaciens and Vibrio spp. are all capable of
reducing trimethylamine oxide to trimethylamine [100].
11
V.P. Lougovois and V.R. Kyrana
The sulfur-containing compounds are perhaps most important as spoilage odor
components, and many bacteria identified as specific spoilers have been shown to produce
them. S. putrefaciens generate hydrogen sulfide from L-cysteine, and methyl mercaptan and
dimethyl sulfide from methionine; some Vibrionaceae also produce hydrogen sulfide [101-
103]. The appearance of hydrogen sulfide-producing bacteria at levels in excess of 10
6
CFU
cm
-2
seems to signal spoilage [104]. On the other hand, neither Pseudomonas nor P.
phosphoreum form significant amounts of hydrogen sulfide [62]. Pseudomonas spp. produce
a number of volatile aldehydes, ketones, esters and non-hydrogen sulfides, imparting fruity,
rotten and sulfydryl odors and flavors [105]. Diacetyl, acetoin, and isovaleric, isobutyric and
acetic acids are of importance in detecting spoilage by B. thermosphacta [60]. Several
spoilage organisms, including S. putrefaciens, P. phosphoreum and Pseudomonas spp., are
known to produce hypoxanthine from inosine or inosine mono-phosphate [57].
Table 1. Specific spoilage organisms of chill-stored fish and off-odor/off-flavor
compounds produced during spoilage
Specific spoilage
organism
Product Spoilage compounds Substrate
Shewanella
putrefaciens
Ice-stored fish
Vacuum-packed
chilled (≤ 4 °C) fish
TMA TMAO
H
2
S Cysteine
CH
3
SH, (CH
3
)
2
S Methionine
Hx Inosine, IMP
Pseudomonas spp. Ice-stored fish Esters, ketones,
aldehydes,
non-H
2
S sulfides
Amino acids
(glycine, serine,
leucine, methionine)
Hx Inosine, IMP
Photobacterium
phosphoreum
CO
2
-packed and
vacuum-packed
chilled (≤ 4 °C) fish
TMA TMAO
Hx Inosine, IMP
Although many psychrotrophic bacteria involved in fish spoilage are proteolytic, it is a
consistent view that proteolysis is not a process of major significance in the early stages of
bacterial spoilage. It has been demonstrated that most of the bacterial proteinase production
and activity occurs during transition from exponential to stationary phase [106]. Thus,
proteolysis of fish muscle normally occurs after spoilage is well advanced; at that stage,
bacterial populations are generally high (10
8
-10
9
CFU g
-1
) and sensorial evidence of spoilage
is already apparent [60]. There is evidence that the presence of free amino acids at a high
level suppresses synthesis of proteolytic enzymes by bacteria. The protein-sparing action of
amino acids may be accounted for by their ability to provide carbon and energy sources for
the spoilage bacteria [79]; the primary mode of utilization is oxidative deamination, which
explains the accumulation of ammonia and volatile fatty acids.
The catabolism of proteins is believed to be essential for survival and growth of the
microflora after low molecular weight compounds are utilized [107]. When bacterial
12
Freshness Quality and Spoilage of Chill-Stored Fish
densities are great and proteinase synthesis is not repressed, as the amino acids have been
utilized, bacteria which are capable of degrading large protein molecules liberate
extracellular proteinases causing breakdown of tissues. During advanced stages of spoilage,
the proportion of proteolytic bacteria to the total aerobic count in the muscles of cod may be
about 30%; thus bacteria may play a significant part in protein hydrolysis in evidently
spoiling fish [5]. As spoilage proceeds, proteolytic degradation becomes more vigorous. The
increased supply of amino acids resulting from proteolysis supports greater production of
ammonia and volatile acids in the latter stages of spoilage. Several volatile sulphur-
containing compounds and indole, skatole, putrescine and cadaverine are also produced from
the bacterial degradation of proteins.
LIPID DETERIORATION
After death, fish lipids are subject to two major changes, namely hydrolysis and
oxidation. Of the two processes, oxidation is the most important, particularly in the
deterioration of frozen fish products, imparting changes in flavor, color, texture and
nutritional value [108]. The reactions involved in lipid oxidation and hydrolysis are either
non-enzymic or catalyzed by microbial enzymes or by intracellular and/or digestive enzymes
of the fish itself [109]. To what extent these reactions will affect the spoilage profile will
depend on such factors as type of fish, maturity, season (metal, lipid and tocopherol levels),
the section of fish, oxygen partial pressure and storage temperature.
Oxidation
Lipid oxidation is a rather complex process whereby unsaturated fatty acids reacting
with molecular oxygen via a free radical chain mechanism, form unstable fatty acyl
hydroperoxides, generally called peroxides or primary products of the oxidation. The
primary auto-oxidation is followed by a series of secondary reactions, which lead to the
degradation of the lipid and the development of oxidative rancidity [110]. The process is
initiated when a labile hydrogen atom is abstracted from a site on the fatty acyl chain, with
the production of a free lipid radical which reacts rapidly with oxygen to form a
peroxyradical. The peroxyradical abstracts a hydrogen atom from another hydrocarbon chain
yielding a hydroperoxide and a new free radical which can perpetuate the chain reaction
[111].
The decomposition of lipid hydroperoxides involves further free radical mechanisms and
the formation of several low molecular weight volatile compounds, some of which have
distinct aromas and can affect flavor properties at concentrations well below 1 ppm. These
breakdown products causing rancidity include complex mixtures of aldehydes, ketones,
alcohols, small carboxylic acids, alkanes, esters, furans and lactones [112]. The rancid off-
odors and off-flavors derived from lipid oxidation may render the product unacceptable or
reduce its storage life. Free radicals and peroxides will destroy vitamins A, C, and E, and
carbonyl products may react with cystine, methionine, tryptophane and lysine to decrease
protein quality through Maillard reactions and give rise to undesirable yellow-brown
13
V.P. Lougovois and V.R. Kyrana
discoloration [113, 114]. In addition, certain oxidation products are potentially toxic [115,
116].
Various tissue constituents may catalyze the oxidation process; these include transition
metals, heme-proteins and enzyme systems associated with both mitochondria and
microsomes [117]. The native anti-oxidant systems in fish tissue are of two types. The first is
represented by enzymes which can remove reactive oxygen species, such as superoxide,
hydrogen peroxide and lipid peroxides, and include superoxide dismutase, catalase and the
peroxidases [118]. The other type is generally represented by low molecular weight free
radical scavengers, such as α-tocopherol located in the lipid interior of the membranes [119],
ubiquinol [120], carotenoid pigments (salmonid fish), and the water-soluble ascorbate and
glutathione peroxidase [108]. Nucleotides have been reported to be inhibitory at
concentrations higher than equimolar in vitro; thus, lipid oxidation would be inhibited in the
early post-mortem period. On the other hand, respiring mitochondria may reduce the amount
of oxygen available for oxidation [117]. It has been suggested that several compounds may
both accelerate and inhibit oxidation, depending on their concentration and association with
other constituents in their proximity [121].
Lipid oxidation can be a major cause of quality deterioration during refrigerated and
frozen storage of finfish, due to the highly unsaturated nature of the fatty acid moieties in
fish tissue [117]. Marine oils typically contain large amounts of long-chain polyunsaturated
fatty acids, with up to six double bonds. Thus, even though most commercially marketable
fish have low fat content, the degree of unsaturation is much higher than in meat, making
fish lipid much more susceptible to oxidation. Rancidity is particularly important at
temperatures below 0 ºC, where much of the free water in the tissue is effectively removed
by crystallization, thereby concentrating catalytic salts and reactants [122]. In fatty species
such as herring and mackerel, which possess lipid reserves in the flesh, lipid oxidation
becomes the dominant spoilage mechanism during frozen storage. In lean fish such as cod
and haddock (0.5-1.1% lipid), around 65% of the total lipids are found intracellularly as
phospholipids associated with the membranes; these too will oxidize, although somewhat
more slowly, and as such will contribute to the deterioration during frozen storage [123]. The
toughened texture, poor flavor and unappealing odor of poorly stored frozen seafood has
been attributed to the binding of oxidized unsaturated lipids to proteins, a process by which
insoluble lipid-protein complexes are formed [124].
By contrast to the frozen storage where lipid oxidation can be the major spoilage
process, the occurrence of rancidity in chill-stored fresh fish has seldom been considered a
problem [125]. Between 0 °C and ambient temperatures, lipid oxidation proceeds 5-10 times
slower in fish muscle than in the extracted oil [122]. Even phospholipids appear to oxidize
slowly at chill temperatures, despite their high degree of unsaturation. This is probably
caused by the highly ordered physical disposition of the lipids, their restricted mobility and
close association with neighboring non-lipid molecules (e.g. proteins) which may interfere
with the oxidation chain reaction [123]. Access to oxygen is another important factor; lipids
embedded in tissues through which oxygen may diffuse only with difficulty will oxidize
more slowly than the exposed lipid surfaces of fillets, minces and gutted fish [117, 126, 127].
In addition, due to competition for oxygen between bacteria, enzymes and lipids, the internal
tissues in whole fish tend to be oxygen deficient and unless the lipids are exposed on the
surface, they will oxidize only slowly [128-130]. It is interesting to note in this context that
14
Freshness Quality and Spoilage of Chill-Stored Fish
factors suppressing bacterial growth (e.g. high CO
2
levels) will allow rancidity to develop if
oxygen is present (Figure 2a).
Lipid oxidation will proceed much more rapidly in the fatty species, the lipids being in
greater concentration and less dispersed through the tissue. With non-fatty fish stored in the
round or as fillets, lipid oxidation is generally not as significant a problem, since
deterioration by microbial action usually occurs before chemical changes are significant
[131]. Rancidity may become a problem in non-fatty fish, which are minced, because of the
incorporation of oxygen into the tissue or the disruption and intermixing of tissue
components. In wet fish storage, lipid oxidation does not appear to be a dominant spoilage
process; components introduced primarily by bacterial spoilage as well as by enzymic
reactions contribute more to the flavor than those derived from lipid auto-oxidation [123,
132, 133], even though in some species such as jack mackerel and mullets, which undergo
rapid quality changes during iced storage, rancid flavors have been reported to affect
acceptability and limit storage life [134, 135].
Differences in susceptibility to oxidation between species may arise from the presence
of higher concentrations of natural antioxidants in fish lipid as well as from a lower
proportion of unsaturated fatty acids in the depot lipids. This would provide an explanation
as to why certain fatty species such as trout, sardines, and gutted mackerel will oxidize at
chill temperatures [136, 137] while others, such as herring, remain relatively unaffected
[133]. Marine finfish are more prone to lipid oxidation than fresh water fish, as they possess
higher levels of the more unsaturated C
20
and C
22
fatty acids on the average. On the other
hand, small or butchered fish are generally more subject to oxidation, presumably because
oxygen availability is not rate-controlling [123].
The origin of the lipid in fish tissue has an important effect on the rate of oxidation
(Figure 2b). In intact fillets, skin lipids have been reported to oxidize more rapidly than the
lipids of dark muscle, which in turn oxidize more rapidly than the lipids of ordinary muscle
[124, 126, 138]. In addition, lipids in the tissue along the visceral cavity will oxidize faster
than lipids in the flapless fillets [139, 140]. The primary difference in the stability of these
lipids is not due to the composition of the fatty acids in the various fractions, but rather to
differences in the quantities and sub-cellular locations of pro-oxidants and anti-oxidants
found in the tissues [117]. The dark muscle has the most unfavorable composition with
respect to lipid oxidation, due to the abundance of heme-proteins, trace metals, microsomal
enzymes, fat and phospholipids; however, dark muscle is fairly well protected by the skin
and white muscle within the intact fillet [126]. Lipids in the skin and the subcutaneous layer
are particularly susceptible to oxidation because of their closer contact with lipoxygenases
[141, 142] and atmospheric oxygen. Trace metals are also a critical factor in skin lipid
oxidation [143]. White muscle has been reported as the most stable, which is in accord with
its low level of pro-oxidative activity [108].
Oxidation potential is greatly influenced by the surface area of the lipid exposed to the
aqueous phase. Lipids found in membrane systems appear to have surface areas that are 50
to 100 times greater, on a weight basis, than those of neutral lipids found in oil droplets
[117]. This is very important in both lean white fish where the principal lipid components are
those found in the membranes and in the dark muscle of fish where mitochondria may
constitute a great part of the volume of the cell [144].
Susceptibility to oxidation may be affected by season and this has been attributed to
either physiological changes (stage of maturity) or changes in diet due to migration [145]. In
15
V.P. Lougovois and V.R. Kyrana
some fatty species, minimum susceptibility to oxidation occurs during post-spawning periods
of heavy feeding, when both total lipid content and the proportion of unsaturated fatty acids
in the depot fat are high; in other fish the converse holds but the reasons for this are not
known [123]. In cultured fish, increased levels of dietary fish oil and digestible protein may
increase the susceptibility of muscle to lipid peroxidation [146]. Diets supplemented with
vitamin E as anti-oxidant appear to preserve lipid quality [147].
0
2
4
6
8
10
12
0 2 4 6 8 10 12
T ime of storage (days)
TBA-RS
0
1
2
3
4
5
0 2 4 6 8 10 12 14 16 18
Days in ice
TBA-RS
Figure 2. Rancidity development in cultured gilthead sea bream (Sparus aurata). (a): Fillets stored at
4 ºC; (■) aerobic storage, (□) modified atmosphere packaging (40% CO
2
- 30% O
2
- 30% N
2
). (b):
Whole, uneviscerated fish stored in melting ice; (■) ordinary (white) muscle, () red muscle, (□) belly
flap. TBA-RS: Thiobarbituric acid-reactive substances (mg malondialdehyde kg
-1
flesh).
During iced storage several changes affecting lipid oxidation can take place, including
activation of heme-proteins [148], increase in free iron [121] and membrane disintegration
leading to large losses of anti-oxidants [120]. Primary processing operations can drastically
alter access to oxygen, thereby affecting rate of oxidation. It has been suggested that the time
lapse between harvesting and gutting/filleting should be long enough to allow for blood
coagulation, while the storage period until further processing/freezing should not exceed two
days, to minimize pro-oxidative changes [108]. Filleting fresh fish under water can improve
storage stability of the fillets, since blood is removed and the access to oxygen is diminished
[149]. Keeping the skin on for as long as possible during storage will protect the sensitive
subcutaneous fat layer and the dark muscle. Storage stability can also be improved by deep-
skinning, involving removal of the skin along with the subcutaneous fat layer and as much
dark muscle as possible [117]. Reductions in yield from this operation will depend very
much on fish species.
The relative importance of lipid oxidation in the spoilage process is complicated by the
fact that many products from fatty species, e.g. herring, may be distributed chilled, having
been processed from fish originally frozen [68]. The practice of freezing the fish before
processing aims at killing parasitic nematode worms (Anisakis species). However, freezing
and thawing can increase the amount of low molecular weight iron and copper released on
storage, thus rendering frozen-thawed fish muscle more susceptible to oxidation than non-
frozen tissue [150].
16
(a)
(b)
Freshness Quality and Spoilage of Chill-Stored Fish
Hydrolysis
Lipid hydrolysis is a common post-mortem feature in fish and fish products. It occurs
through the intervention of endogenous and/or bacterial lipolytic enzymes, the major
products being free fatty acids (FFA) and glycerol. Free fatty acids are virtually absent in the
fat of living animal tissue, but can form after the animal is killed. Phospholipids are
hydrolyzed most readily, followed by triacyglycerols, cholesterol esters and wax esters [5].
Lipid hydrolysis of chill-stored eviscerated fish is of minor importance; considerable
FFA concentration may, however, develop during storage of whole, round fish, particularly
at elevated temperatures [109]. In the fattier species, triacylglycerols in the depot fat are
cleaved by triglyceride lipase originating from the digestive tract or excreted by certain
microorganisms. Lipolysis can be more profound during the later stages of storage, due to a
greater diffusion of digestive enzymes from the viscera of spoiling fish, as well as to the
intervention of bacterial lipases [128, 129]. In lean fish such as cod and haddock, the
enzymes responsible for the production of free fatty acids are believed to be cellular
phospholipases, with non-enzymic reactions and the bacteria present on the chilled fish
playing negligible roles [151, 152]. However, a correlation between the activity of these
enzymes and the rate of lipolysis has not, as yet, been established [109]. Phospholipase
release from lysosomes may be triggered by enzymic and non-enzymic peroxidation [153].
Because of the polyunsaturated type of the fatty acids bound to phospholipids at
glycerol-carbon atom 2, the hydrolysis may lead to increased oxidation, since free fatty acids
are more susceptible than fatty acids esterified to glycerol. However, uncertainty still
surrounds the relationship between lipolytic activity and oxidation. Some authors have
reported that lipid oxidation occurs more rapidly in tissue containing FFA [154], while
others have shown that FFA produced from phospholipids inhibit lipid oxidation in muscle
tissue [155].
The consequences of lipolysis on acceptability of fish and fish products are still not so
clear. The fatty acids themselves may cause a “soapy” off-flavor. However, although the
terms rancid and soapy are often used as descriptors in taste panel score sheets of chilled and
frozen stored fish, no correlation appears to have been established between the development
of rancidity or soap-like flavors and fatty acid production [123, 128, 156, 157]. The fatty
acids and/or soaps produced from lipid hydrolysis may interact with proteins, thereby
affecting textural qualities of the fish [158].
CHANGES IN SENSORY ATTRIBUTES DURING SPOILAGE
Sensory attributes are those perceived with the senses, i.e. appearance, odor, texture and
flavor. The characteristic sensory changes in fish post-mortem vary considerably with
species, season, catching method, fishing ground and storage conditions, and virtually reflect
the autolytic, microbial and chemical deterioration processes that render the fish unfit for
human consumption.
17
V.P. Lougovois and V.R. Kyrana
Changes in Appearance
Freshly caught fish have a shining, iridescent skin covered with a thin layer of uniformly
spread, nearly transparent slime. The eyes are bright, convex, with jet-black pupil and
transparent cornea. The gills are generally bright pink or red and free from visible slime. As
the fish spoils, the skin loses its bloom and smooth feel and becomes dull, bleached, and
rough to the touch. The eyes gradually shrink and pass from being flat to concave (sunken),
the pupil becoming cloudy and milky and the cornea opaque. The gills assume a bleached,
light pink appearance that finally changes to yellowish or grayish brown, and the slime on
skin and gills becomes turbid, clotted and discolored, as a result of increased bacterial
growth [159].
The flesh of fresh fish has a translucent appearance as a result of the incident light
scattering reflectively and equally. Melting ice in contact with the fish permits water to
penetrate the skin and reduce translucency. While spoilage progresses the flesh becomes
opaque because the incident light is unevenly scattered due to the gradual disintegration of
myofibrils and their wider and more random intracellular distribution [113]. The color of the
white fish flesh changes from light creamy to gray and the peritoneum becomes dull and can
be progressively more easily detached from the internal walls of the visceral cavity.
The problem of discoloration is one of the most serious concerns of the seafood industry.
In general, with ground fish it is desirable to have a fillet that is uniformly white and this is
best achieved by properly bleeding the fish before it dies [160]. During spoilage of intact
fish, the blood contained in the kidneys, its associated vessels and main artery along the
backbone gradually diffuses into the adjacent flesh causing blood discoloration. Rough
handling may also cause bruises, rupture of blood vessels and blood oozing into the tissue.
Depending on the extent of occurrence of blemishes and defects such as bloodstains,
bruising and discoloration, the fish may become unacceptable for some purposes like retail
display of wet fillets [159].
The important pigments for red color in salmon, sea trout and tuna are carotenoids
(primarily astaxanthin) and heme-proteins (myoglobin and hemoglobin). The presence of
astaxanthin has a direct influence in the red color of salmonid fish flesh [161, 162].
However, fresh water and marine fish cannot synthesize carotenoids de novo [163]; these
pigments are derived from dietary sources. The accumulation of astaxanthin and other fat
soluble carotenoids in the muscle may function as a co-factor in the post-mortem antioxidant
process [164, 165], as well as a depot for pigments needed at the time of spawning when the
male develops a strong red color in the skin and the female transports carotenoids into the
eggs [166]. Post-mortem oxidation of the carotenoid pigments results in fading of the pink or
red color of fish flesh and skin. A lypoxygenase-type enzyme present in skin tissue can
convert astaxanthin into colorless carbonyl compounds at refrigerated temperatures in the
dark, while a myeloperoxidase found in fish leukocytes appears to cause rapid discoloration
of β-carotene in the presence of hydrogen peroxide and iodide or bromide ions [113].
Discoloration in tuna fish results from the oxidation of red myoglobin and oxy-
myoglobin of the dark muscles to brown met-myoglobin, the rate of oxidation being
dependent on the temperature of storage. Other forms of discoloration may also occur,
including the greening of tuna and the yellowing of the flesh of species like mullet [73]. The
greening phenomenon has been attributed to the oxidation of an exposed cysteine residue of
myoglobin by trimethylamine oxide [2]. Yellow discoloration of mullet flesh may develop as
18
Freshness Quality and Spoilage of Chill-Stored Fish
a result of the migration of carotenoid pigments from chromophores or caroteno-protein
complexes in the skin to the subcutaneous fat layers. Lipid oxidation and carbonyl-amine
reactions may also result in yellowing of fish flesh.
Changes in Odor/Flavor
The mild, delicate flavors and aromas of very fresh fish are contributed by volatile six,
eight, and nine-carbon aldehydes, ketones and alcohols arising from the action of specific
lipoxygenases on long-chain polyunsaturated fatty acids [41, 167]. The six-carbon volatile
compounds hexanal, (Z)-3-hexenal, and (E)-2-hexenal occur mainly in freshwater fish; they
are formed by a 15-lipoxygenase acting on n-3 or n-6 polyunsaturated fatty acids and
provide distinctly green plant-like aromas [168]. Eight-carbon volatile alcohols and ketones,
e.g. 1-octen-3-ol, 1-octen-3-one, (Z)-1,5-octadien-3-ol, (Z)-1,5-octadien-3-one, are found in
most finfish where they contribute heavy plant-like and metallic-like flavors. These
compounds require the action of a 12-lipoxygenase and a lyase which forms the vinyl
alcohol; a dehydrogenase is then involved in the formation of the corresponding ketone [41].
Nine-carbon aldehydes, such as (E,Z)-3,6-nonadienal, (E,Z)-2,6-nonadienal and (E)-2-
nonenal, can be formed from the eicosapentaenoic acid by a 12-lipoxygenase in conjunction
with a lyase; these volatiles contribute fresh, green cucumber-like flavors and aromas [169-
171]. The carbonyl compounds appear to dominate, their thresholds being 10,000 times
lower than those of the corresponding alcohols.
In marine fish these green, planty, and melony flavor notes are usually accompanied by
sea-, brine- or iodine-like flavor notes, provided by volatile bromophenols, which are
biochemically-formed by marine algae, sponges, and bryozoa, and become distributed
through the food chain. Very low concentrations (ng/g) of 2-bromophenol, 4-bromophenol,
2,4-dibromophenol, 2,6-dibromophenol and 2,4,6-tribromophenol have been shown to
impart salt-, shrimp-, crab- and iodine-like flavors (172-174]. Excessive levels may,
however, lead to defective chemical, phenol-like or iodoform-like flavors [175].
Bromophenols have been reported to be virtually absent from cultured fish because of
feeding practices and this could account for some of the differences in flavor between prime
wild and cultured fish [176].
Certain 5'-mononucleotides, mainly adenosine mono-phosphate and inosine
monophosphate, are also known to have an important impact on fish flavor. These
compounds are strongly flavorful constituents of fish flesh, which enhance or modify the
flavor of other compounds, particularly in combination with glutamic acid [177]. The
concentration of inosine monophosphate occurring in fish muscle stored in ice for one day
after death is at least ten times that required to cause flavor enhancement [178] and it is
conceivable that the sulfide flavor-suppressing properties of the mononucleotide could be
advantageous in some circumstances.
In chill-stored fresh fish, the delicate aromas and flavors are often soon lost through
autolytic degradation of non-volatile taste-active nucleotides and microbial conversion of
fresh, plant-like aroma volatiles [e.g. (E,Z)-2,6-nonadienal] to less intensely flavored
compounds [e.g. (E,Z)-2,6-nonadienol]. The fresh odor gets weaker and changes from green,
cucumber-like to sweet, flat and insipid, due to the increase in alcohol fractions and the
presence of short-chain esters [179]. In fatty species, carbonyl compounds appear to
19
V.P. Lougovois and V.R. Kyrana
dominate all the time [180, 181]. After a period where the odor is described as neutral or
non-specific, the first indications of off-odors are detectable. Initially, compounds having
grassy, fruity, slightly sour and acidic notes (musty, milky, bready, malty and beery) are
formed. These will progressively change in character and become sour, acetic and strong
fishy, due to the accumulation of volatile fatty acids and amines. Trimethylamine is known
to intensify fishiness by a synergistic action with unsaturated volatile aldehydes [182].
Lipoxygenases in the gills and skin act on fresh fish aroma compounds providing
hydroperoxides; as a result, fishy, oxidized odours develop at an accelerated rate in these
regions. During the later stages of spoilage, stale cabbage-like, sulfurous, hydrogen sulfide
and rancid odors develop, while in the putrid stage the character becomes ammoniacal and
fecal [159]. Of the wide variety of volatile auto-oxidation products occurring in rancid fish,
the 2,4,7-decatrienals derived from long-chain n-3 polyunsaturated fatty acids in fish lipid
appear to be the most important contributors to fishy, cod liver oil-like odours [41]. Spoiled
or putrid smells are caused by the presence of sulfur compounds, phenols, certain fatty acids
and (Z)-4-heptenal [183]. Indol, putrescine, cadaverine and other diamines derived from
bacterial degradation of amino acids also give rise to putrid smells. The odors of the external
surfaces and gills or internal organs, where these are present, are more intense than those
developing in the flesh.
A closely-related sequence of changes occurs in the flavor of fish flesh; the sweet,
creamy and meaty flavors typical of fresh fish weaken on storage, until the flesh becomes
insipid, flavorless, and finally sickly sweet, sulfurous, rubbery, bitter and rancid. In iced fish,
leaching of flavor compounds in the ice-melt water enhances the general reduction in flavor
observed during the early stages of storage, but may also have the opposite effect of slightly
improving quality by reducing the concentration of undesirable flavors in spoiled fish [159].
The pattern of change in odor and flavor is broadly similar for all white finfish, but
details differ, depending on the species and origin of the fish. While this undoubtedly reflects
variations in intrinsic composition and enzymic activity, it is conceivable that these
differences also select different strains in the flora to grow [68]. It is evident from sensory
evaluation that the spoilage of marine temperate-water fish is characterized by the
development of offensive fishy, rotten, H
2
S off-odors and off-flavors, while for some
tropical marine fish and fresh-water fish, fruity and sulfydryl off-odors and off-flavors are
more typical [57]. Fatty fish differ distinctly from lean fish because characteristic flavor
changes result from the degradation of lipids.
Textural Changes
Fish muscles contain a large number of highly active enzymes and their catheptic
activity is much greater than that of the mammalian muscle. Connective tissue content is
generally lower in fish than in meat animals and the cross-links formed by fish collagens are
less extensive [184]. Resolution of rigor mortis is also more rapid in fish than in terrestrial
animals. All these factors contribute to an enhanced rate of fish flesh softening during chill
storage [185]. Thus, unlike meat, the post-mortem tenderization of fish muscle is highly
undesirable for processors and consumers [186]. The deteriorative changes in fish muscle
texture involve loss of firmness and springiness, and increase in softness, causing the spoiled
fish flesh to become mushy. A paste-like texture can be sometimes encountered in the
20
Freshness Quality and Spoilage of Chill-Stored Fish
muscles of fresh fish (e.g. South American hake, Merluccius sp.) which have been strongly
infected by protozoan parasites (Myxosporidea) or in salmon during spawning migration,
due to enhanced protease activity [72, 187].
The progressive post-mortem tenderization or softening of fish muscle tissue has been
attributed to weakening of Z-discs of myofibrils [188], changes in the molecules of
connectin [189], weakening of the myosin-actin junction [190], and alterations in the
pericellular connective tissue [191, 192]. Most of these processes result from the post-
mortem activities of endogenous proteases on myofibrilar proteins; calcium-dependent
proteases and the lysosomal cathepsins D and L appear to bring about most of the textural
changes in chill-stored fish, since they are quite active in the pH range of 5.5 to 6.5 [73].
Collagenases may also be involved in the process. It has been suggested that cleavage of
non-helical regions and/or cross-links of type V collagen molecules causes disintegration of
collagen fibrils and weakening of the pericellular connective tissue, thereby leading to
softening of fish muscle [192, 193]. Fish skeletal muscle contains several alkaline proteases
[194-196], but the role of these enzymes in texture deterioration appears to be important only
at elevated temperatures [197].
Gaping is a major textural quality defect, originating from the rupture of thin tubular
sections in the myocommata. This undesirable state is generally associated with fish that
resumed feeding after a period of starvation, particularly in early summer, but occurs also in
farmed fish [198, 199]. The mechanical strength of the connective tissue that holds the fillets
together is strongly influenced by post-mortem pH. The myocommata are strong at neutral
pH but greatly weakened at more acid values such that fillets gape. Pre-harvest exercise may
induce gaping-related changes in the fine structure of the myocommata-muscle fiber junction
[200]. Because of their damaged appearance, fillets that have gaped are difficult to sell.
Furthermore, they cannot be mechanically skinned, hung on a tender for smoking or sliced
[7]. The incidence and severity of gaping can be reduced significantly by early filleting
[201].
FACTORS AFFECTING STORAGE STABILITY
Several factors may influence spoilage and potential storage life of fresh fish. These
include species, fishing ground and season, water temperature, method of harvesting and
immediate post-harvest handling, storage conditions and fishing vessel sanitation. The
fishing industry has very little control over intrinsic features of the fish (e.g. size, physical
condition and composition) that impact upon their perceived quality [159]; further, it is not
always possible to be selective in the harvesting methods utilized for many commercial
species. It is, therefore, critical that the fish be handled in a quality conscious manner as soon
as they are landed on the vessel [54].
21
Table 2. Descriptive terms related to changes in sensory attributes of whole,
round gilthead sea bream (Sparus aurata) stored in melting ice
Days
in ice
Skin Outer slime Eyes Gills (appearance)
Gill and internal
odors
Raw texture
Flesh and gut cavity
appearance
0
3
Bright; iridescent;
metallic silver gray
sheen;
well differentiated
colors
Glossy; thin;
transparent
Bulging; convex lens;
black bright pupil;
translucent cornea
Convex lens; black
pupil with slight loss
of initial clarity
Glossy, bright
pink or red; clear
mucus
Fresh; iodine; sea
weedy; shellfish
odors
Flesh firm and
elastic; body pre-
rigor or in rigor
Less sharp sea
weedy and shellfish
odors
Body setting off
rigor mortis
White flesh with bluish
translucency; cut surfaces
stained with blood; glossy
gut cavity; peritoneum
strong, well adherent; blood
bright red, flows readily
6
10
Loss of brilliance of
color; very slight
bleaching
Aqueous;
transparent
Slight flattening of
plane; loss of
brilliance
Loss of gloss and
brightness
Freshly cut grass;
week sea weedy
Plane; slightly gray
pupil; slight opacity
of cornea
Slight loss of
color; clear mucus
Slight musty,
mousy, milky
Flesh firm and elastic
to the touch
White flesh; some loss of
bluish translucency;
peritoneum slightly dull, well
adherent; blood less bright,
becoming dark and
coagulated
14
17
Dull with some
bleaching; some loss
of scales
Opaque and
somewhat
milky
Plane or concave;
slight opacity and
reddening of cornea
Bleached with
some brown
discoloration and
cloudiness of the
mucus
Musty; lactic; slight
sour; boiled
cabbage
Some softening of
the flesh but finger
indentations not
retained
White flesh; loss of bluish
translucency; slight
yellowing of cut surfaces of
belly flaps; peritoneum dull,
easily torn from flesh
Muddy; putrid Softening of the
flesh; finger
indentations slightly
retained
Waxy appearance of flesh
but no reddening;
21 Loss of
differentiation and
general fading of
colors; overall dull
grayish
pigmentation
Yellowish-
gray; clotted
Concave to sunken;
gray pupil; opaque,
red cornea
Brown or
bleached; mucus
yellowish-gray
and clotted
Fecal; amines;
sour; acidic;
sulfides
No elasticity; flesh
flaccid and sticky
Some browning of kidneys
and blood; peritoneum weak,
very easily torn
It has been recognized for some time that amongst marine fish there are differences in
spoilage rates. This is in part due to differences in chemical composition, but other factors are
also involved. In some species such as rainbow trout and winter flounder, antimicrobial
activity has been demonstrated in the slime [202, 203]; the skin of plaice, but not that of cod,
possesses a powerful lysozyme [204]. Also, many fish caught in tropical waters are generally
less prone to rapid spoilage and exhibit a longer refrigerated storage life than their cold-water
counterparts, although the dominant bacterial types are similar at the time when spoilage is
evident [62]. Cold-water fish species harbor an established psychrotrophic flora, which is not
inhibited as effectively by refrigeration as are the normal bacteria of tropical water fish. Such
flora would decrease the lag time before outgrowth and subsequent spoilage under
refrigerated or iced storage. The endogenous enzymes from cold temperature-acclimated fish
would be expected to enhance autolytic spoilage to a greater extent than those derived from
tropical or warm-water species [73]. On the other hand, psychrotrophic organisms naturally
occurring on fish harvested from warm and tropical waters have an intrinsically lower growth
rate at chill temperatures [22].
Physical stress during capture can adversely affect post-harvest quality and storage life of
the fish. Tuna harvested by purse seining may become very excited and die in a highly
stressed state. In conditions impairing oxygen supply, the ensuing strenuous struggling just
prior to death may induce extreme glycolysis and acidosis in the muscles [5]. The rapid
decline in muscle pH combined with elevated body temperature results in symptoms of burnt
tuna; the flesh is no longer bright red but appears pale, muddy brown or turbid with soft
texture, and the flavor is acidic with a metallic aftertaste [54, 73]. This is similar to the pale,
soft, exudative (PSE) condition that may develop in pork. Rapid built-up of lactic acid in the
muscle of yellowtail flounder that have struggled excessively during harvesting leads to an
abnormal condition known as “chalk”, wherein the flesh exhibits a dry, white chalky
appearance and fibrous texture [205]. The effect of physical stress on post-harvest quality has
been documented also with rainbow trout [206] and salmon caught by gill netting [207];
bacterial infection increases with stress and earlier signs of deterioration occur in fish
struggled to death. In chub mackerel, exhausting struggling promotes the collapse of collagen
fibrils in the endomysium, thereby accelerating muscle softening [208].
Several countries require that certain ground fish be bled, gilled and eviscerated on board
the fishing vessel, before packing in ice. Bleeding can result in a more attractive white
appearance of the fillet, although there is some question whether it actually benefits quality in
all situations [54]. The bleeding of trawled cod can be beneficial to quality only if conducted
within 1-2 hours of the fish being brought aboard, the benefits varying with location of catch
and time of year [209]. Disagreement exists as to the cutting method. In cod-like species,
bleeding is normally accomplished by severing the arteries just behind the gills and in front of
the heart; a deep throat cut including the dorsal aorta can be beneficial. There seems to be
general agreement that best bleeding is achieved while the fish is still alive or at least in a
pre-rigor state, as it is the muscle contractions that force the blood out of the tissues [210].
Since most of the blood is located in the internal organs, bleeding must be followed by
immediate evisceration and thorough washing to reduce numbers of contaminating bacteria.
Very often, bleeding and evisceration are performed in one step through the removal of the
viscera, which also provides a reasonable degree of bleeding [205].
V.P. Lougovois and V.R. Kyrana
2
3
4
5
6
7
8
9
10
0 2 4 6 8 10 12 14 16 18 20 22
Days in ice
TVC (cfu/g)
0
5
10
15
20
25
30
35
40
45
0 2 4 6 8 10 12 14 16 18
Days in ice
k
1
value (%)
Figure 3a. Changes in total viable counts (20 ºC)
on standard plate count agar, during iced storage
of whole (■) and eviscerated (□) sharpsnout sea
bream (Diplodus puntazzo). Means of two fish.
Figure 3b. Regression lines for k
1
value against
time of iced storage in whole (■) and eviscerated
(□) sharpsnout sea bream. Means of three fish.
Average RSD, 8.45% (whole fish), 8.80%
(eviscerated fish).
3
4
5
6
7
8
9
10
0 2 4 6 8 10 12 14 16 18 20
Days in ice
Sensory score for flavor
3
4
5
6
7
8
9
10
11
12
13
0 10 20 30
Days in ice
GR-Torrymeter reading
Figure 3c. Changes in freshness score for flavor
of cooked fillets from whole (■) and eviscerated
(□) sharpsnout sea bream stored in ice. Means of
five trained panelists’ assessment. Average RSD
7.64% (whole fish), 7.79% (eviscerated fish)
Figure 3d. Regression lines for Torrymeter
readings against time of iced storage in whole
(■) and eviscerated (□) sharpsnout sea bream.
Means of six fish. Average RSD 9.20% (whole
fish), 10.3% (eviscerated fish)
Viscera are a source for spoilage, particularly when the fish have been feeding heavily.
Products of bacterial metabolism, digestive enzymes and fecal material can diffuse through
the stomach and the intestinal walls and permeate the flesh, causing discoloration, off-flavors
and even belly bursting in uneviscerated fish [22]. Gutting the fish within a short period after
catching may serve to retard autolytic spoilage processes as well as removing a large reservoir
of potential spoilage bacteria [140, 211]. However, the cutting opens flesh surfaces to direct
24
Freshness Quality and Spoilage of Chill-Stored Fish
bacterial attack and renders the visceral cavity more susceptible to oxidation and
discoloration. Contamination of the exposed flesh resulting from direct and/or environmental
transfer (gut and skin bacteria, contaminated knives, etc.) can limit the storage life of
eviscerated fish [212]. As a result, there continues to be controversy concerning the
advantages and disadvantages of gutting at sea.
Work on farm-raised sharpsnout sea bream (Diplodus puntazzo), cultivated in net cages
in sea water and starved for 3-5 days prior to slaughter by hypothermia, revealed that gutting
had no significant effect on quality or storage life (Figures 3a-d). In this study, samples were
subjected to sensory evaluation, flesh bacterial load tests, GR-Torrymeter measurements, and
determination of nucleotide metabolites (k
1
value) and trimethylamine nitrogen levels. In all
cases no significant differences were observed between whole and eviscerated fish. Storage
life, determined by sensory evaluation of cooked fish flavor, was between 15 and 16 days in
both groups. Although Torrymeter measurements and k
1
value tented to indicate a somewhat
faster deterioration rate in the eviscerated fish, the differences were not significant.
The conflicting results in the literature lead to the conclusion that the gutting procedure
should not be applied universally in all harvesting operations. Factors such as species and size
of the fish, amount of lipid, fishing ground and method of harvest, should be taken into
consideration before deciding whether or not evisceration can be beneficial to quality and
storage life.
On-board processing must not subject the fish to lengthy delays in chilling. Controlling
the temperature of newly caught fish is perhaps the most important element in maintaining
quality and extending storage life. Fish should be cooled down to the temperature of melting
ice as quickly as possible, and this is normally achieved through the direct application of
flaked ice or through the use of chilled sea water (CSW) or refrigerated sea water (RSW).
Delays in chilling, particularly when ambient temperatures are high, can significantly shorten
shelf life during subsequent refrigerated storage [80, 213, 214]. Holding the fish at high
ambient temperatures of 28-30 °C, even for short periods (9-12 hours) before icing, enables
mesophilic organisms (e.g. Bacillus spp.) to multiply rapidly and play a more important role
in the spoilage [82].
In general, the bacterial counts on temperate-water fish stored in melting ice show a lag
phase from 1 to 5 days, exponential growth from 6 to 14 days, and stationary growth
thereafter; under most commercial conditions, freshness quality deteriorates rapidly from 12
days onward [22]. On the other hand, the bacterial flora on tropical fish chilled immediately
after capture and held near 0 ºC show some extension of the lag phase and a marked decrease
in growth rate. As a result, long storage lives (> 25 days) have been observed in a number of
warm-water species [215, 216].
Although broad differences are observed in the storage lives of various finfish, the effect
of temperature on relative spoilage rate is very similar in all fresh fish products; increasing
the temperature from 0 ºC to 5 ºC doubles the rate at which fish spoil, while an increase to 10
°C can enhance spoilage by a factor of 5-6, significantly reducing storage life (Figure 4).
Since it is a common practice to store fresh fish in melting ice, a reference temperature of 0
ºC is normally used when comparing storage times. Relative spoilage rates (R) in the
temperature (T ºC) range from 0 ºC to 25 ºC can be calculated from the equation [217]:
R = (0.1T + 1)
2
25
V.P. Lougovois and V.R. Kyrana
Where the temperature history of a fish after capture is known, changes in freshness
quality may be estimated by integrating the relative spoilage rates with time, resulting in an
equivalent length of storage time at 0 ºC [218]. This allows spoilage models to be used for
predicting the effect of varying storage temperatures on product shelf life, but assumes that
the fish is in prime condition upon harvesting, and that handling is conductive to good fish
quality. Poor handling would result in much more rapid spoilage than the equivalent “length
of time on ice” would indicate.
2
4
6
8
10
12
14
0 1 2 3 4 5 6 7 8 9 10 11
St orage t em perat ure (degrees Celcius)
Storage life (days)
y = 0.104x + 0.966
R
2
= 0.962
0.8
1
1.2
1.4
1.6
1.8
2
2.2
0 1 2 3 4 5 6 7 8 9 10 11
St orage t em perat ure (degrees Celcius)
R
1/2
Figure 4. Influence of storage temperature on shelf life (left) and relative rate of spoilage R (right) of
aerobically stored gilthead sea bream (Sparus aurata) fillets
Inadequate chilling is a major factor contributing to histamine (or scombroid) fish
poisoning (HFP), a food borne chemical intoxication caused by eating spoiled, or bacterially
contaminated, fish [219, 220]. The finfish most commonly associated with HFP appear to
have naturally high levels of free histidine in the muscle [221], and include tuna, bonito,
mackerel and dolphin-fish (mahi-mahi). Scombroid toxins presumably arise from the
bacterial production of histamine and other biogenic amines via decarboxylation of the parent
amino acids. Several bacteria have been incriminated with the production of biogenic amines
in fish muscle [222-226]. Members of the Enterobacteriaceae, specifically Morganella
morganii, certain strains of Klebsiella pneumoniae and a few strains of Hafnia alvei are the
most prolific histamine producers in fish maintained at temperatures greater than 4 °C [227].
Species of Photobacterium and Vibrio may be primarily responsible for histamine production
at low temperatures [228-230]. Although some of the histamine-producing bacteria are
naturally present in the microflora of marine fish, most seem to arise from handling during
and after harvesting. Once the decarboxylases are formed, their activity will continue, albeit
at a slower rate, even if subsequent storage temperatures do not support bacterial growth [227,
231].
Rapidly chilling newly caught fish and maintaining a low temperature (≤ 2 ºC) during
subsequent storage is also critical for preventing or delaying the growth of a wide range of
marine and terrigenous pathogens that potentially may be present on finfish after initial
processing, including Vibrio spp., Salmonella spp., Staphylococcus aureus, Bacillus cereus,
E. coli, Cl. perfringens, L. monocytogenes, and non-proteolytic Cl. botulinum [22].
26
Freshness Quality and Spoilage of Chill-Stored Fish
Strict sanitary control may not benefit the quality of well-iced whole fish, even though it
is still important to avoid cross contamination. However, when primary processing operations
are conducted on-board the fishing vessel, an unsanitary environment will expose the
practically sterile flesh of newly caught fish to high numbers of bacteria, thereby contributing
to reduced quality and storage life [54].
EVALUATION OF FISH FRESHNESS QUALITY
Quality Terminology
Quality is not a specific object that can be measured directly; it is a complex concept
implying different thinks to different people, depending on socioeconomic status, education
and life-style, age, tradition, etc. [232]. Most often quality refers to the aesthetic appearance
and freshness or degree of spoilage that the fish has undergone. However, the exact definition
of quality rests ultimately with the buyer or consumer. In the fish processing industry quality
often relates to expensive species or to the size of the fish. Fish considered by a processor to
be of inferior quality may be too small or in too poor a condition for a certain process,
resulting in low yield and profit [72]. The consumers often think that best quality is found in
fish consumed within the first few hours post-mortem. However, fish in rigor are difficult to
fillet and skin and are often unsuitable for smoking; slightly older fish that have passed
through rigor mortis are, thus, more desirable for the processor. Also, newly caught fresh tuna
may be of poor quality due to acidosis in the muscle induced by exertion. On the other hand,
to government authorities that are mainly concerned with health hazards, good quality means
the absence of harmful bacteria, parasites or chemicals [14]. Accordingly, the definition of
quality is deliberately a wide one because different aspects are important in different
circumstances. Botta [20] defines quality as the degree of excellence to which a product
meets all of the attributes, characteristics and features that the buyer or consumer of the
product, and the regulatory agencies expect”.
In trying to assess fish quality, a number of variables should be taken into consideration,
including safety (chemical and microbiological), intrinsic composition and nutritional value,
freshness or degree of spoilage, availability, yield and profitability, convenience and integrity
[233]. Although safety is indisputably the most important type of fish quality, the buyer or
consumer is generally not capable of readily determining if the fish is indeed safe. Similarly,
the buyer or user cannot determine nutritional quality. For those attributes that affect health
and safety, the government or other independent official bodies are expected to safeguard
consumers’ interests by the enforcement of standards or drawing up of regulations [159].
However, freshness quality defined in terms of specific sensory attributes of the fish (e.g.
appearance, odor, flavor, and/or texture) is consciously or unconsciously assessed each time a
product is being consumed, and is therefore an extremely important factor in determining
overall quality of fish [20].
27
V.P. Lougovois and V.R. Kyrana
Methods of Assessing Freshness Quality
The state of freshness can be described by a variety of clearly defined attributes of the
fish, which can be assessed and quantified by various indicators [234]. A great number of
methods have been proposed for assessing fish freshness in industry, but few have found
practical application [235, 236]; many have proved to be unsuitable for the purpose because
of their inaccuracy and impracticability, and others are only useful for research or product
development. It is often convenient to describe methods of assessing freshness quality as
sensory and non-sensory or instrumental. The latter category comprises numerous chemical,
biochemical, physical and microbiological methods.
Sensory assessment employs the same senses used by consumers when they make
subjective judgments about quality, and is therefore a more secure way of obtaining
information about freshness quality than is non-sensory assessment [237]. Human senses are
better at recognizing complexities and can be more discriminatory than instruments.
However, their responses can vary, particularly with fatigue or outside distraction, and using a
panel of trained assessors can be expensive and inconvenient [159]. Non-sensory assessment
is based mainly on measuring major physical or chemical alterations from the original
condition of the fish. Among the advantage of non-sensory methods are their convenience,
normally lower cost, and the ability to set quantitative standards. An ideal instrumental
method should be fast, reliable, consistent with sensory assessment, and preferably applicable
to all seafood; it should correlate freshness quality with the time and temperature of storage
following the harvest, and should provide a basis for estimating future storage life. However,
this is very difficult to achieve in view of the variety of species and biochemical changes [5].
Non-sensory methods usually require laboratory facilities and trained staff and are
necessarily destructive. In addition, they measure only one aspect of spoilage or may even
assess some change in the fish not directly related to spoilage. Thus, non-sensory assessment
is only secondary and cannot ideally substitute measures of quality as understood by
consumers [238]. This is because instruments and analyses cannot yet replace human senses.
Non-sensory methods can appear more objective and reliable and less variable than sensory
methods, although this need not be the case; reduced variability means that non-sensory
methods are more precise, not necessarily more accurate. Therefore, arbitrarily classifying all
instrumental methods of evaluating freshness quality as being objective and all sensory
methods as being subjective is now widely recognized as wrong [20]. However, courts of law
may find it easier to accept the results of chemical or physical tests, being based on impartial
instrumental readings, than the results of sensory tests [238].
Sensory Methods
Sensory evaluation may be defined as the scientific discipline used to evoke, measure,
analyze and interpret human reactions to characteristics of food as perceived through the
senses of sight, smell, taste, touch and hearing [239]. Sensory methods are, thus, wholly
dependent upon the human senses, perhaps aided occasionally by simple devices like a ruler.
In order to have a complete picture of freshness quality, each of appearance, texture, odor and
flavor must be measured [160]. Although considerable advances are being made in the
development of instruments that can measure individual quality changes, most sensory
28
Freshness Quality and Spoilage of Chill-Stored Fish
characteristics can only be measured meaningfully by humans [14]. Thus, freshness
assessment of fish, though often monitored by physical, chemical and bacteriological
methods, necessarily rests finally on sensory evaluation.
Sensory methods are of two kinds, subjective and objective. In the former, biases in
judges are not minimized and personal opinion is allowed free rein [240]. The subjective
methods are affective tests based on a measure of preference or acceptance. They are applied
in market research and product development and essentially test the consumer’s attitude and
emotional response to the product [239]. In objective sensory methods, biases are deliberately
minimized by the use of specially trained expert assessors with a high degree of sensory
sensitivity and experience in sensory methodology. Trained experts have been taught to
function as analytical instruments and are able to make consistent and repeatable assessments
[20]. The objective sensory methods can be divided into discriminative and descriptive.
Discriminative testing (triangle test, ranking, paired comparison) is used to find small
detectable differences between samples, which may arise from changes in ingredients,
processing method, packaging and storage conditions. For example, paired comparison may
be used to detect increases in the toughness of fish stored in the frozen state against unfrozen
controls; triangle tests are useful in determining if ingredient substitution gives a detectable
difference in a product, and ranking enables the determination of the threshold for detection
of oil taints in fish [240]. Descriptive testing is, however, by far the most commonly used
method for assessing freshness quality of chilled fish. Descriptive tests (structured scaling,
quality index method, profiling) can be used to determine the nature and intensity of existing
differences [14].
Since grading the freshness quality consists of sorting fish into specified categories, the
defined grade standards, which describe each category, are extremely important. Stale or
putrid fish are easily recognized by sight, smell or taste, and freshness assessment of fish in
this condition presents little difficulty. There are, however, many occasions when it is
necessary to assess fish at some intermediate stage of loss of freshness or deterioration.
Consequently, the selected terms used to describe an individual grade of a single criterion
must be precise, technically correct, objective, and independent [20, 241].
During the past few decades many scoring systems were developed for sensory analysis
of raw fish. The most extensive scheme, but not necessarily the best operationally, is that
developed at Torry Research Station, Aberdeen, Scotland, fifty years ago [242]. The scheme
is based on discriminatory descriptive changes in the attributes of appearance, odor, flavor
and texture of the raw and cooked fish, each change being denoted by a number. Odor and
flavor are allowed a maximum score of ten, five being the maximum for the remaining
attributes. The fundamental idea was that different attributes changed independently and not
necessarily in step. An important feature of the Torry scheme is that over most of the scale the
scores are linearly related to the length of time the fish are held in ice. Modifications
involving the pooling of attributes have been introduced into the scheme to make it more
useful for industrial use [237].
In Europe, the most commonly used method for quality grading of chilled fish at the
point of first sale is the EU scheme [243]. In this, three grades of freshness are laid down (E,
A and B) corresponding to various stages of spoilage; E (Extra) is freshest fish, whilst below
B is the level where fish can not be marketed and by implication are not fit for human
consumption. Initially, the EU scheme was based on only a few major food fish, but has now
been extended to a much greater range of species. However, the scoring system has not yet
29
V.P. Lougovois and V.R. Kyrana
been modified to take account of detailed species differences. In the EU scheme, a more or
less continuous scale of changes has been segregated into a few distinct stages for the purpose
of defining grades of freshness quality. As a result, the scheme gives no information about
remaining shelf life of the fish, but is useful whenever the quality of batches of fish has to be
assessed rapidly, such as at port markets or factory reception areas [159]. The inspector scans
the fish taking into account, more or less simultaneously, a number of different attributes
before allocating a grade.
Table 3. Freshness quality grading system for round European sea bass
(Dicentrarchus labrax)
Attribute being assessed Defined Characteristic Demerit points
SKIN
Appearance Bright, shining, iridescent 0
Loss of iridescence 1
Slightly dull/discolored 2
Odor Fresh 0
Neutral 1
Slight off-odors 2
Spoiled 3
Slime Clear, transparent 0
Slightly opaque 1
Opaque, yellowish gray and sticky 2
EYES
Cornea Clear, translucent 0
Slightly cloudy 1
Cloudy, opaque 2
Pupil Black, bright, shiny 0
Slightly cloudy 1
Gray - white 2
GILLS
Appearance Uniformly bright pink 0
Less bright, slightly faded 1
Faded, discolored 2
Odor Fresh, characteristic 0
Neutral 1
Slight off-odors 2
Spoiled 3
TEXTURE
Body stiffness Very stiff, hard (in rigor) 0
Firm, elastic (finger marks not retained) 1
Some softening (finger marks retained) 2
Total demerit points 0-18
30
Freshness Quality and Spoilage of Chill-Stored Fish
In order to describe the different freshness quality levels in a more precise and well-
documented way, a new rapid scaling method, the Quality Index Method (QIM), has been
developed [244-246]. This freshness quality grading system involves specifying the
characteristics of a number of appropriate sensory attributes of the raw fish. Once the
characteristic of a sensory attribute is determined, it is assigned a demerit score ranging from
0 to 3. The scores for all characteristics are then summed to give an overall sensory score, the
so-called quality index. The scale gives zero score for absolutely fresh fish, while
increasingly larger totals result as fish deteriorate [247, 248]. The maximum score for each
sensory attribute depends on the detectable variability of that attribute, and thereby
determines its relative importance in the total qual