BIOCHEMICAL CHANGES IN

FISH

Shanti Dwita Lestari, Sherly Ridhowati

MK Fisiologi Hasil Perikanan
Penyebab pembusukan ikan
 Aktivitas biologis : bakteri, jamur, ragi, dan
serangga
 Aktivitas enzim (autolisis)
 Fisik akibat proses penanganan
 Kimiawi : adanya reaksi kimia seperti ketengikan
akibat oksidasi lemak dan denaturasi protein
Faktor yang mempengaruhi
kemunduran mutu
 Cara/alat penangkapan
 Cara penanganan
 Reaksi ikan menghadapi kematian
 Jenis dan ukuran ikan
 Keadaan fisik sebelum mati
 Suhu lingkungan
 Kandungan protein dan lemak ikan
 Jaringan otot yang lemah
 Penanganan yang tidak benar dan tidak tepat
Apa yang terjadi Ketika ikan mati
Ketika ikan mati
 Proses mulai dari pre rigor – in rigor – post rigor
terjadi selama 1 – 3 jam atau beberapa hari
setelah ikan mati
Postmortem Stages
 Pre-rigor
 Rigor Mortis
 Post Rigor
Prerigor
 Muscle tissue is soft and pliable
 Active glycolysis ceases: NO Oxygen!
 A fall in ATP and creatine phosphate level
 Glycogen is converted into lactic acid
 pH decreases
 Anaerobic glycolysis

 At the point of death, the supply of oxygen to the muscle

tissue is interrupted, no oxygen is available for normal
respiration
 For most teleost fish, glycolysis is the only possible
pathway for the production of energy once the heart stops
beating.
 Anaerobic glycolysis produces 3 moles (or 2 moles) of ATP
for each hexose unit broken down:
(C6H10O5)n + 3ADP + 3H3PO4 + H2O 2CH3CH(OH)COOH
+ 3ATP + (C6H10O5)n-1
C6H12O6 + 2ADP 2CH3CH (OH) COOH + 2ATP
 Anaerobic glycolysis has principally lactic and pyruvic acids
as its end-products, accumulation of lactic acid which in turn
lowers the pH of the muscle
 ATP is produced in glycolysis, but only 2 moles for each
mole of glucose oxidized as compared to 36 moles ATP
produced for each mole of glucose if the glycolytic end
products are oxidized aerobically in the mitochondrion in
the living animal.
 Thus, after death, the anaerobic muscle cannot maintain its
normal level of ATP
 Due to the hydrolysis of ATP, inorganic phosphate is formed,
which then stimulates the degradation of glycogen to lactic
acid
 When the intracellular level declines from 7-10 µmoles/g to
1.0 µmoles/g tissue, the muscle enters rigor mortis.
Fall in ATP and creatine phosphate level

Aerobic and anaerobic breakdown of glycogen in fish muscle

 For some time after death, ATP is maintained at a
definite level in the muscle by active creatine kinase
which catalyzes the resynthesis of ATP from ADP and
creatine phosphate
Creatine kinase
Creatine Phosphate + ADP Creatine + ATP

 In the early prerigor phase, the concentration of ATP

remains relatively constant, but there is rapid decline
in CP levels
Lohmann reaction

 an easily reversible reaction occurring in muscle, in

which the high
energy phosphate bond of ATP is transferred to
creatine, forming creatine phosphate; the reaction is
catalyzed by creatine kinase.
Drop in pH

pH values and glycogen content of animal tissue

(Sakaguchi, 1994)

Drop in pH (5.1-5.5) causes in inhibition of glycogen

breaking enzyme : DISRUPTION of ATP production
Rigor Mortis
 Stiff and rigid condition
 pH falls and actomyosin complex is formed
 Loss of extensibility proceeds slowly at first (delay
phase) then extremely rapid (fast phase)
 Fish exhibit rigor mortis period commencing 1-7h
after death
Stiffening of the muscle
 Rigor mortis is caused by the bonding of myosin
heads to the active centers of thin filaments, the
actin, which leads to the formation of a rather rigid
structure of inter-connected myofilaments, the
actomyosin.
 When the level of ATP reaches its minimum, myosin
and actin are interconnected irreversibly, resulting in
rigor mortis
 Muscular contraction
 Fish has muscle cells
running in parallel
and connected to
sheaths of
connective tissue
(myocommata),
which are anchored
to the skeleton and
the skin. The
bundles of parallel
muscle cells are
called myotomes
Muscular contraction
 The muscle tissue of fish is composed of striated
muscle.
 One functional unit consists of sarcoplasma
containing nuclei, glycogen grains, mitochondria and
a number (up to 1 000) of myofibrils. The myofibrils
contain the contractile proteins, actin and myosin.
These proteins or filaments are arranged in a
characteristic alternating system making the muscle
appear striated upon microscopic examination
Schematic representation of a sarcomere. The thick and thin filaments overlap in the
region of the A-band, with the I-band formed from the thin filaments only. The central
M-line anchors the thick filaments and the Z-disk the thin filaments. Titin is found along
the length of the sarcomere. Tropomyosin and the troponin complex interact with actin
to form part of the thin filament
 The body musculature of fish is a series of
segmental myotomes of complex shape separated
from each other by collagenous sheets, called
myosepta
 Muscle fibres are orientated at angles up to 40o to
the longitudinal axis and, hence, myotomes have
complex three-dimensional structure (Fig. 1).
Myotomal shape is the deep Wshape and the
orientation of fibres is not random to contract at
about the same rate
Muscular contraction
 Muscle contraction starts when a nervous impulse sets off a
release of Ca + + from the sarcoplasmic reticulum to the
myofibrils.
 When the Ca + + concentration increases at the active
enzyme site on the myosin filament, the enzyme ATP-ase is
activated.
 This ATP-ase splits the ATP found between the actin and
myosin filaments, causing a release of energy which is used
as contractile energy making the actin filaments slide in
between the myosin filaments in a telescopic fashion,
thereby contracting the muscle fibre.
 When the reaction is reversed (i.e., when the Ca + + is
pumped back, the contractile ATP-ase activity stops and the
filaments are allowed to slip passively past each other), the
muscle is relaxed.
Stiffening of the muscle (2)
 Due to the ATPase activity of myosin head during the
formation of actomyosin, ATPs are also depleted
 ATP is one of the most important component for muscle
contraction, functioning both as a fuel for contraction
and as a plasticizer in the presence of Mg++
 During rigor mortis progress, myofribillar ATP is absent,
whereby the actin and myosin filaments stay interlocked
as actomyosin.
 This is why the muscle becomes rigid and stiff during
postmortem rigor mortis.
Post Rigor
 Fish muscle gradually relax again and it becomes
limp, but no longer as elastic as before rigor
 The rate in onset and resolution of rigor varies from
species to species and is affected by temperature,
handling, size and physical condition of the fish
 If the fish is cooked pre-rigor the texture will be
very soft and pasty. In contrast, the texture is tough
but not dry when the fish is cooked in rigor. Post-
rigor the flesh will become firm, succulent and
elastic.
Factors influencing the rigor mortis progress

 Species (fatty fish-lean fish ; dark muscle-white

muscle)
 State of activeness (pelagic fish-demersal)
 Condition and seasonality
 Degree of exhaustion
 Size
 Handling
 Temperature
 stressed fish can show a propensity to rapid drop in
pH, poor color and liquid-holding capacity, gaping
and soft texture
 low pH associated with accumulation of lactic acid
leads to the soft texture, gaping, high cathepsin
activi
 duration of pre-slaughter stress affects the texture,
where short-term stress have demonstrated
softening of fish muscle, whereas the long-term
stress increases the muscle firmnessty and liquid loss
CHANGES IN FISH FLESH BIOCHEMISTRY POST
MORTEM

Autolysis :

• Nucleotide Breakdown
• Protein Denaturation
• Lipid Oxidation and Hydrolysis
• Bacterial Attack
 BIOCHEMISTRY OF GLYCOGEN DEGRADATION

sh /crustacean Vigorous antemortem exhaustion of energy

being caught struggle reserves (glycogen and high-energy
Phosphates)

Degradation of Asphyxia phenomena with gradual

high-energy phosphates formation of anoxial conditions in the
muscle

Production of hypoxanthine degradation of glycogen

Formation of formaldehyde,
ammonia, inorganic phosphate following Embden-Meyerhof-
and ribose phosphates Parnas pathway

rigor Accumulation of lactic acid and

a reduction of pH
mortis
Nucleotide breakdown
In fish, uric acid can be formed by hydrolysis of
IMP or breaking down of Hx due to high
degree of xanthin oxidase activity.
K-value
 The concentrations of ATP, ADP, AMP, IMP, inosine and can be measured
by high performance liquid chromatography (HPLC).
 The ratio of inosine and hypoxanthine to the total amount of the above
substances is represented as a percentage K-value ---Saito et al.(1959)
 K value of less than 20% represents fresh fish, whilst anything greater is
indicative of spoilage.
 sterile Non sterile

The rates of formation and breakdown of IMP were the same in both sterile
and non- sterile samples of cod tissue indicating that the catabolic pathway
for the degradation of ATP through to inosine is entirely due to autolytic
enzymes.
BIOCHEMISTRY OF PROTEIN DEGRADATION

SARCOPLASMIC PROTEINS

 Sarcoplasmic proteins are soluble proteins in the muscle sarcoplasm.

 They include a large number of proteins such as myoglobin, enzymes,
and other albumins.
 Sarcoplasmic enzymes are responsible for quality deterioration of fish
after death and before bacterial spoilage.
 The significant enzyme groups are hydrolases, oxidoreductases, and
transferases
MYOFIBRILLAR PROTEIN DETERIORATION

 The most common myofibrillar proteins in the muscles of aquatic

animals are myosin, actin, tropomyosin, and troponins C, I, and T
 The degradation of myofibrillar proteins in seafood causes these
proteins to lose their integrity and gelation power in icestored seafood
 This situation gets even worse when the proteins are cross-linked due
to the presence of formaldehyde formed from trimethylamine
degradation.
 The cooked products become tough, chewy, and stringy or fibrous
STROMAL PROTEIN DETERIORATION

 The residue remaining after extraction of sarcoplasmic and myofibrillar

proteins is known as stromal protein. It is composed of collagen and
elastin from connective tissues
 the degradation of collagenous matter textural changes
Cathepsins
 cathepsins are "acid" proteases usually found packaged in
tiny, submicroscopic organelles called lysozomes, released
into the cell juices upon physical abuse or upon freezing and
thawing of post mortem muscle
 Cathepsins D and L are believed to play a major role in the
autolytic degradation of fish tissue
 The enzyme was far less active in the presence of ATP,
suggesting that such an enzyme would only be active in post
mortem fish muscle
 Cathepsin L contributes more to 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
Calpains
 "calpains" or "calcium activated factor" (CAF) is grouped in
intracellular proteases and associated with fish muscle
autolysis and is found in meats, finfish and crustaceans
 Calpains have been found primarily responsible for the post
mortem autolysis of meat through digestion of the z- line
proteins of the myofibril
 In crustacean muscle, calpains are associated with
moltinduced textural changes to the muscle and carry out
non-specific generalized digestion of the myofibrillar
proteins
 Fish calpains digest myosin (specifically the myosin heavy
chain) to form an initial fragment with approximate
molecular weight of 150 000 Da
Collagenases
 autolytic collagenase enzymes cause "gaping" or
breakdown of the myotome during long-term
storage on ice or short term storage at high
temperature
BIOCHEMICAL CHANGES IN NONPROTEIN NITROGENOUS
COMPOUNDS

 white meat generally contains less NPN compounds than the dark meat
 in the meat of white fish, the NPN generally made up 9–15% of the total
nitrogen, in muscles of mollusks and crustaceans 20–50%, and in some
shark up to 55%
 95%of the total amount of NPN in the muscle of marine fish and shellfish is
composed of free amino acids, imidazole dipeptides, trimethylamine oxide
(TMAO) and its degradation products, urea, guanidine compounds,
nucleotides and the products of their postmortem changes, and betaines
 The endogenous enzymatic break down TMAO to dimethylamine (DMA)
and then formaldehyde, while the bacterial action reduce TMAO to TMA
 the production of DMA and formaldehyde takes place mainly in anaerobic
conditions
 Trimethylamine oxide (TMAO is present naturally in
many marine animals as an osmoregulator and as a
means of excreting nitrogen
 After death, the sh muscle produces large amounts of
ammonia due to degradation of ATP to AMP followed
by deamination of AMP
 Both ammonia production and degradation of TMAO
can be either endogenous or contributed by bacteria
 Ammonia, TMA, small amounts of DMA, and
methyamine constitute the “total volatile base” an
indicator of freshness commonly used for seafood
TVB - Total Volatile Base
 The amount of TVB is measured by distilling a fish
extract, and determining the base concentration by
titration against acid.
 A fresh sample of Jack Mackerel would have a
value of 19-21mg TVB N/100g, whilst an ageing
sample would be nearer 30 mg TVB N/100g.
Effects of formaldehyde formation on protein

 formaldehyde binds covalently to various functional

groups in proteins and hence results in a
deconformation of the protein, followed by cross-
linking between the protein peptide chains via
methylene bridges
 The interaction of formaldehyde with muscle protein
accelerates muscle protein denaturation
 The presence of formaldehyde also causes a
noticeable decrease in the extractability of total
proteins, particularly the myobrillar group
BIOCHEMICAL CHANGES IN LIPID

 Aquatic animals are especially rich in long-chain PUFAs which are

found in higher numbers in the membrane phospholipids than in
storage triglycerides
 phospholipids are more prone to lipid oxidation due to their higher
level of unsaturation and their significantly larger surface area
compared with neutral lipids more exposure to a variety of
prooxidants (such as heme proteins, radicals, iron, and copper)
 These prooxidants, especially the heme proteins hemoglobin and
myoglobin, can lead to high levels of lipid oxidation products,
especiallyas pH of the muscle is reduced (which occurs postmortem)
 Lipid Oxidation and Hydrolysis

Lipids degrade by two mechanisms :

 the oxidation of fish oils yielding the rancid odours and tastes which are the
major problem encountered in fish storage
 the enzymatic hydrolysis of lipids (fats) to produce free fatty acids and
glycerol
Lipid oxidation assessment

Hydroperoxides Value
 Oxidation leads to rancidity, the degree of which is commonly evaluated by
measuring the free fatty acid and peroxide concentrations.
 Hydroperoxides can be measured by mixing the fish oil with potassium
iodide, and measuring the amount of iodine liberated by titration against
thiosulphate.
 The hydroperoxides oxidise the iodide to iodine, which is liberated
according to the following equation:
Thiobarbituric acid (TBA)

 A further measure of oxidation is the TBA test.

 This involves extracting some fish muscle into trichloroacetic acid and
treating it with with thiobarbituric acid (TBA).
 The TBA reacts with malonaldehyde, a substance formed during oxidation,
to form a red compound.
 The intensity of the red colour, which is proportional to the concentration of
the malonaldehyde, can be measured using a spectrophotometer.
BIOCHEMICAL CHANGES IN PIGMENTS

heme proteins Red meat

(warm-
(hemoglobin and myoglobin) blooded) sh

cold-
pigments in blooded shellsh
seafood
hemocyanin
such as crustaceans
and mollusks

carotenoids shellsh
products
EPITHELIAL DISCOLORATION

 Ommochromes : epidermal chromatophores in squid

 In prerigor squid, the pigment is dispersed throughout
the chromatophores: hence the dark red-brown
appearance of the epithelial tissue
 Following rigor mortis, the pigment cells contract, the
continued storage of squid results in a structural
deterioration of the ommochrome membrane, leading to
bleeding of pigment and downgrading of quality : red
discoloration of the meat, sub-cutaneous yellowing of
esh below the pigmented skin
HEMOGLOBIN
 Hemoglobin contributes less to the appearance of
seafood than myoglobin because it is lost easily during
handling and storage, while myoglobin is retained in the
intracellular structure.
 The amount of residual hemoglobin in sh muscle is
inuenced by the bleeding efcacy at the time of catch
and the method of catch.
 The green meat of raw or frozen swordsh (Xiphias
gladus) is believed to be due to the combination of
hemoglobin with hydrogen sulde generated from the
fairly extensive decomposition of the meat
HEMOCYANIN
 Hemocyaninsarecopper-containingproteins, as
compared with iron-containing proteins in
hemoglobins and they combine reversely with
oxygen.
 Blue discoloration of canned crabmeat is associated
with a high content of hemocyanin.
 The average copper content of blue meat (e.g.,
2.8mg%) is higher than meat of normal color (e.g.,
0.5mg%)
MYOGLOBIN

 Myoglobin in fish muscle is retained in the intracellular structure.

 The myoglobin content in muscle of yellowfin tuna was found to range from
37 to 128 mg% in the light-colored muscle and from 530 to 22,400 mg% in
the dark-colored muscle
 Myoglobin in fish is easily oxidized to a brown-colored metmyoglobin.
 The discoloration of tuna during frozen storage is associated with the
formation of metmyoglobin, depending on temperature and location
 Greening is a discoloration problem arises from the formation of a sulfhydryl
adduct of myoglobin in the presence of an oxidizing agent
(e.g.,trimethylamine oxide, TMAO).
MELANINOSIS (MELANIN FORMATION)

 Melaninosis (melanin formation) or “blackspot” in

shrimp during postmortem storage is caused by
phenol oxidase
 This problem can be overcome by the use of
appropriate reducing or inhibiting agent(s).
Quality Index Method (QIM)
 Quality Index Method (QIM) is a freshness grading
system and most convenient method for rapid
salmon quality and freshness assessment and
estimation of the remaining storage time in ice
(Sveinsdottir, Martinsdottir et al. 2002). The QIM
scheme is developed for various species
 Pseudomonas and Altermonas putrefaciens are
probably the major bacterial species that cause
fundamental spoilage of usually iced fish. These can
use the non-protein nitrogen compounds present in
the fish such as trimethyl amine oxide (TMAO) that
result in several volatile odoriferous compounds such
as trimethyl amine oxide, TMA (Regenstein and
Regenstein 1991). These volatile compounds are
responsible for the off-odours and off-flavours
characteristic of spoiled fish.
 Bacteria are able to decompose proteins, other nitrogen containing
compounds to ammonia, hydrogen sulphide, which produce an
unpleasant and disgusting flavour (Herbert and Shewan 1975).
Trimethyl amine oxide (TMAO), mostly found in marine fish, is broken
down to trimethyl amine (TMA), dimethyl amine (DMA) and ammonia
(NH3), which are responsible for off-odours in fish undergoing
spoilage. The main spoilage test of metabolite(s) produced during
fish storage or distribution to obtain a quantitative fish quality index
is total volatile bases (TVB). It measures the total content of
TMA+DMA+ ammonia plus other basic nitrogenous compounds
associated with fish spoilage. TVB and TMA values of 30 mgN and
15 mgN/100 g are the rejection spoilage levels respectively
(Regenstein and Regenstein 1991). The fishy odour of TMA when it
reacts with lipid is generally detectable when the TMA level reaches
4-6 mgN/100 g.
 Chemical spoilage processes are changes taking
place in the lipid fraction of the fish. Lipids are
oxidised to peroxides, aldehydes, ketones and
lower aliphatic acids. The hydro-peroxides are
tasteless but can cause brown and yellow
discolouration of the fish tissue. The degradation of
hydro-peroxides gives rise to the formation of
aldehydes and ketones that result in rancid off-
flavours. All the chemical by-products eventually
reach a level where the fish is rejected