Fish processing

1. Changes in fish after death, spoilage of fish,
spoilage and pathogenic microorganism.
Lecture - 2

2. 1. Pre-rigor condition:
After death, the muscle is totally relaxed and the limp elastic texture
usually persists for some hours.
Physiologically, after death aerobic oxidation stops and anaerobic
oxidation of glucose takes place, which leads to the formation of
lactic acid and pH of muscle decreases.
 This process takes place till glycogen is completely used up.
Total process of rigor mortis can be divided into three phases/
conditions:
Level of phosphoric acid increases, enzyme creatine kinase
completely utilizes ATP and leads to next step rigor mortis condition.
At this stage, thick and thin filaments of myofibriller proteins are free
to slide on each other, which indicates that muscle are extensible and
can contract on stimulation.

3. Under anaerobic conditions, ATP may be synthesized by two other
important pathways
• Creatine phosphate - restricted to vertebrate muscle (teleost fish)
• Arginine phosphate - some invertebrates such as the cephalopods
(squid and octopus)
ATP production ceases when the creatine or arginine
phosphates are depleted
octopine is the end-product from the anaerobic
metabolism of cephalopods and is not acidic (unlike
lactate)
After death, when the regeneration of ATP ceases, the ATP is rapidly
degraded i.e. after death, the anaerobic muscle cannot maintain its
normal level of ATP, and when the intracellular ATP level declines
from 7 - 10 µmoles/g to <1.0 µmoles/g tissue, the muscle
enters rigor mortis.

4. 2. Rigor mortis:
 At the final phase of ATP breakdown, myosin crosses bridges,
interaction is establishes firmly between the thin and thick
filaments (i.e. actin and myosin), thereby making it non-extensible
and hence non-contractile.
 Actin and Myosin combine in the presence of calcium ions and
forms actomyosin.
 Resolution of the rigor is a slow process essentially due to the low
pH-favored hydrolysis of actomyosin by acid proteases such as
cathepsin that are present in the muscle.
 Cathepsin is the most important enzyme responsible for protein
degradation in muscle and internal organs and converts them into
amino acids.

5. 3. Post-rigor conditions: It includes the real spoilage of fish due to
autolysis and microbial spoilage. Autolysis is also known as self-
digestion, controlled by enzymes.
Changes in fish muscle during different phases of postmortem
events

6.  After death pH initially declines (pre-rigor phase) then become
constant (rigor phase) and then increases (post rigor phase).
 All ATP exhausts during pre-rigor phase.
 During pre-rigor phase Ca++ ion increases and during rigor phase it
becomes constant.
 Bacterial invasion occur during post rigor phase only.

7. Microbial spoilage:
Composition of the microflora on newly caught fish depends on the
microbial contents of the water in which the fish live.
Microbial growth and metabolism is a major cause of fish spoilage
which produce
• Amines
• Biogenic amines such as putrescine, histamine and
cadaverine, organic acids, sulphides, alcohols, aldehydes and
ketones with unpleasant and unacceptable off-flavors.

8. CHANGES IN FISH AFTER DEATH, SPOILAGE OF FISH,
SPOILAGE AND PATHOGENIC MICROORGANISM
Fish Spoilage and Quality
Fish spoilage starts as soon as the fish dies. In tropical countries, fish
spoils quite rapidly, within few hours of landing if not properly
cooled.
In raw fish, spoilage takes place mainly due to enzymatic action,
chemical action and microbial action.
Enzymes and bacteria do not cause any deteriorative changes in the
living cell because of natural defensive mechanism.
In dead fish, enzymes become involved in autolytic changes and
bacteria can invade the fish muscle and proliferate there.
Fish gut is rich in proteolytic enzymes and in dead fish it digests the
gut and belly region making the fish very soft.

9. Factors responsible for spoilage:
1. Water content (internal water content and humidity): Fish
consists of on average 70% water; in fatty fish this percentage is
about 65% and in lean fish about 80%. With such high levels of
internal moisture, bacteria can grow rapidly. Meat forms a
protective layer on the flesh as a result of drying out at low
humidity. A film of condensation is formed on cold meat lying in
warm surroundings, which is a good medium for bacteria and
moulds.
2. Oxygen content: Strictly aerobic micro-organisms need oxygen for
their growth, while strictly anaerobic micro-organisms can only
grow in the absence of oxygen. For example, minced meat spoils
very quickly because a lot of air has been mixed into it.

10. 3. Acidity: Acidity of a product is indicated by its pH. Fish and meat
have a neutral pH, i.e. 7. Bacteria only grow between a
minimum pH of 4.5and a maximum of 8-9 with an
optimum of 6.5-7.5. As a result, fish very susceptible to
spoilage. When fish is fermented, the pH is deliberately
kept low so that only the desired microorganisms affect
the product and not those bacteria which cause spoilage.
4. Specific chemical composition: Bacteria need sources of energy
and nitrogen. Minerals and vitamins are also important
for growth. In meat, the first source of energy used by
bacteria is sugar, then lactate, free amino acids and only
then protein. Sources of nitrogen are nitrate, ammonia,
peptides, amino acids or products of decomposition.

11. 5. Temperature
Temperature for the growth of micro-organisms is between 7 °C and 55
°C (45-131o F). With freezing, micro-organisms are inactivated, and
with long-term heating all micro-organisms will eventually die. At
temperatures above 80°C (176o F) they usually die. Spores are often
resistant to temperatures above 100°C (212oF).
Apart from all these preconditions for growth, the time between
contamination and processing or consumption is also of importance.
Some micro-organisms grow faster than others. At 37 °C (99oF) certain
bacteria can multiply from 1,000 to 10,000,000 individual organisms in
seven hours. The actual rate at which bacteria grow depends on a
combination of the factors mentioned above.

12. Signs of spoilage: - Off odour and off flavours, slime, gas production,
discoloration and soft texture.
Components involved in spoilage process are protein, lipids,
Carbohydrates, nucleotides and other non-protein nitrogen
compounds.
Rate of spoilage is temperature dependent & lowering the temperature
will reduce the rate of spoilage.

13. Autolytic spoilage: - Autolytic spoilage is responsible for early loss of
quality of fresh fish.
 First change occurs in fish muscle by the gradual hydrolysis of
glycogen to lactic acid, known as glycolysis.
 Second change occurs in the form of nucleotides degradation.
 Third enzyme change is due to the reactions of digestive
enzymes.

14. Reactions of Glycolysis: - After death, the blood circulation stops and
cells are no longer supplied with oxygen & hence glycogen
will not be converted into CO2 & water, now glycolysis
proceeds via the anaerobic pathway where the end product is
lactic acid, which lowers the pH of muscle.
Glycolysis continues until supply of glycogen is completely used up.
Glycogen is more concentrated in the dark muscle than in the white
muscle.
In stressed fish glycogen is rapidly depleted.

15. Nucleotide degradation:- Nucleotide degradation in fish muscle
produces many flavour bearing compounds. These
compounds are formed by the splitting of ATP by a series of
dephosphorylation and deamination reactions.
ATP → ADP → AMP → IMP → Inosine + Ribose → Hypoxanthine
Reactions of digestive enzymes:
Digestive enzymes causes belly bursting in fish, especially during a
period of high food intake.
Such fish will degrade quickly and spoil easily, at the same time
bacteria proliferate & produce gases such as CO2 and H2.
This gas production leads to belly bursting.

16. Reactions of polyphenol oxidose:
Development of black spot of shrimp is due to the presence of an
enzymes polyphenol oxidose (PPO).
Black spot (melanin) is also formed by the oxidative reaction
(oxidation) of tyrosinase on tyrosine.
This pigment is formed on the internal shell surfaces or in advance
stage shrimp meat also. It makes the shrimp unattractive for
marketing. Shrimp phenol oxidase differs from that found in
mussel or lobster that it is not activated by trypsin.

17. Treatment:
Dip treatment in 0.2 - 0.5% sodium bisulphate for one minute can
prevent black discoloration resulting due to phenolase reaction.
In Crab, phenolases cause blue-black coloration. In crab, an enzyme
is present in blood, which is responsible this blue black colouration.
Dip treatment of citric acid, ascorbic acid, sodium bisulphate
and EDTA prevents from enzymatic reaction.
Haemocyanin induced blacking is also prominent in crab and is non-
enzymatic.

18. Discolouration:-
Yellow discolouration:-Frozen storage of some fish may result in
yellowing of flesh below the skin. Freezing or other processes disrupt
chromatophores and release carotenoids & their migration to the sub-
cutaneous fat layer causes yellowing.
Yellowing is associated with lipid oxidation and carbonyl-amine
reaction during frozen storage.
Brown discolouration:-Brown discolouration (yellow discolouration
also) is caused by the reaction of protein or amino acids with product of
lipid oxidation.
Discolouration due to protein-lipid browning is greater in fatty fish than
lean fish.

19. Enzyme(s) Substrate Changes Encountered Prevention/lnhibition
glycolytic
enzymes
Glycogen - production of lactic
acid, pH of tissue drops,
loss of water-holding
capacity in muscle
- high
temperature rigor may
result in gaping
- fish should be allowed
topass through rigor at
temperatures as close
to0°C as practically
possible
- pre-rigor stress must be
avoided
autolytic
enzymes,
involved in
nucleotide
breakdown
ATP
ADP
AMP
IMP
-loss of fresh fish
flavour, gradual
production ofbitternes
with Hx (later stages)
-same as above
- rough handling or
crushing accelerates
breakdown
Cathepsins Proteins,
peptides
- softening of tissue
making processing
difficult or impossible
- rough handling during
storage and discharge
Summary of Autolytic Changes in Chilled Fish

20. chymotrypsi
n,
trypsin,
carboxy-
peptidases
Proteins,
peptides
- autolysis of visceral
cavity in pelagics
(belly-bursting)
- problem increased with
freezing/thawing or long-
term chill storage
Calpain Myofibril
lar
proteins
- softening, molt-
induced softening in
crustaceans
- removal of calcium thus
preventing activation?
collagenases Connecti
ve tissue
- "gaping" of fillets
- softening
- connective tissue
degradation related to time
and temperature of chilled
storage
TMAO
demethylase
TMAO - formaldehyde-
induced toughening
of frozengadoid fish
- store fish at temperature <
-30°C
- physical abuse and
freezing/thawing accelerate
formaldehyde-induced
toughening

21. Spoilage due to Chemical Reactions:-
Fish lipid contains high level of polyunsaturated fatty acids
(PUFA) which undergoes oxidative changes. Particularly with fatty
fish, fat oxidation gives/develops rancid (off) flavour and odour as
well as discolouration.

22. Lipid oxidation occurs due to two reasons:
•Auto oxidation- Action of O2 on the unsaturated fatty acids.
•Lipid hydrolysis- An enzymatic hydrolysis with free fatty acids
(FFA).
In pelagic species like Sardine, Mackerel ad Herring, rancidity
has been detected during spoilage. During spoilage first
hydroperoxides are formed, which further degrade to form aldehyde
and ketones with typical rancid flavour.
Process of oxidation is initiated and accelerated by heat, light (UV-
radiation), presence of several organic or inorganic compounds (eg Cu
and Fe), moisture content, large surface, and presence of air etc.
Antioxidants such as tocopherol, ascorbic acid, citric acid, carotenoids,
BHA, BHT, PG, IAG, NDGA etc can inhibit oxidation.
butylated hydroxy anisole) and BHT (butylated hydroxy toluene Propyl gallate, Isoamyl gallate,
Nordihydro guaiaretic acid

23. Microbial spoilage:- Fish spoilage is mainly due to the action of
bacteria. Bacteria are present on the surface slime, skin, gills and
intestine of fish. In dead fish, bacteria began to invade the tissues
causing spoilage and production of undesirable compounds. Such
bacteria on the fish are very much depending on the microbial flora of
the environment.
Types of bacteria:
Spoilage type bacteria
Pathogenic bacteria
Non-indigenous bacteria

24. •Spoilage type bacteria:
These bacteria are responsible for the spoilage of fish. Important class
of spoilage organisms found in tropical species is Pseudomonas,
Flavorbacter, Acinetobacter, Aeromonas ad Moraxella.
Spoilage bacteria are characterized by their ability to produced H2S,
reduce trimethylamine oxide (TMAO) to trimethylamine (TMA) and
convert Urea to Ammonia. Pseudomonas produces many volatile
sulphur compounds.
Fish flesh starts visibly to spoil when bacterial level rises above
107cfu/g. Flesh loses its culinary qualities like juiciness, firm texture
etc. During spoilage, product become soft with loss of flavour,
discolouration and off flavour.

25. Major deterioration due to microbial activities are:
Ammonia:- Spoilage organisms convert many nitrogen compounds
into off smelling volatile bases. Non-protein compounds present in fish
are good substrate for spoilage organisms. Free amino acid pool in the
muscle of fish is readily utilized by typical spoilage organisms by the
process of deamination. These results in the formation of Ammonia
which is major component in the total volatile nitrogen (TVN) [TVN
also use as indicator for fresh fish]. Urea present in elasmobranches like
sharks and rays is degraded to ammonia by bacterial action.
Trimethyl amine (TMA):- In marine fish, an odourless compound
called trimethylamine (TMAO) is present. Marine flat fish &teleosts
contain low levels of this compounds (0.1-0.5%), while elasmobranches
(shark, rays etc) and gadoids contain very high levels (1 - 1.5%).
Spoilage bacteria convert this substance into foul smelling
trimethylamine (TMA). This reaction is temperature dependent. Fishy
odour is produced when it reacts with fat.
TMAO TMA

26. Histamine:- Microbial spoilage of fish produces the toxin, histamine
in certain fishes that causes histamine poisoning or scombroid
poisoning. Scombroid fishes and other dark muscle fish contain high
level of free amino acid i.e. histidine in their muscle. During spoilage
histidine is converted to histamine by bacteria. Tuna, Bonito,
Mackerel, Blue fish, Dolphin fish (Mahi mahi) Carangids, Herring,
Sardine and anchovies shows more frequently histamine poisoning.
37oC is optimum temperature for microbial activity Morganella
morganii, Klebsiella pneumoniae and Hafnia alvei are the main
spoilage organisms producing histamine. Low temperature storage
right from catch reduces histamine production.

27. Indole:-Conversion of tryptophan to Indole is another result of amino
acid decomposition by bacteria. FDA uses indole level along with
sensory evaluation for measurement of shrimp decomposition.
Other compounds formed during bacteria spoilage:- Free amino
acids, sugars, peptides, creatine along with lipid and protein also
produced by bacteria spoilage. Important volatile sulphur compounds
such as hydrogen sulphide (H2S), dimethlysulphide (CH3)2S and
methylmercaptan (CH3SH), easters of lower fatty acids such as acetic,
propionic, butyric and hexaenoic acids are also produced. Volatile
sulphur compounds influence the organoleptic characters, especially
odours in spoiling fish.
Some of the spoilage bacteria are proteolytic and produce ammoniacal
odour by producing ammonia from the protein breakdown.

28. Compounds produced by bacterial action:-
Substrate Compounds
•Inosine Hypoxanthine
•Methionine and cysteine Acetic acid, CO2& H2O
•Methionine and cysteine H2S, CH3SH and (CH3)2S
•Tryptophan Indole
•Glycine, Leucine and Serine Esters of acetic, propionic,
butyric and hexaenoic acid
•Urea Ammonia
•Lipids Carbonyls
•Protein Tyrosine, Indole, Skatole,
putriscine, cadaverine
•Histidine Histamine

29. Pathogenic bacteria:-
E.g. Clostriclium botulinum, Vibrio sp Aerononas sp.
Pathogenic bacteria are widely distributed in environment and not a
serious problem in fresh fish but they grow and multiply in processed
food during storage and cause illness when consumed.
Non-indigenous bacteria:-
E.g. Salmonella sp. Shigella sp. E coli and Staphylococcus
aureus
It comes in processed fish through contamination. Source include
polluted aquatic environment, sewage, excreta from animals, birds,
human beings, workers handling the material as well as the surface &
environment where the fish/shell fish is processed.

30. Bacteria of public health significance associated with fish and
shellfish are given below:
•Salmonella spp
•Shigella spp.
•Enteropathogenic Escherichia coli
•Staphylococcus aureus
•Clostridium perfringens
•Aeromonas spp.
•Clostridium botulinum
•Vibrio parahaemolyticus
•Vibrio vulnificus
•Vibrio cholerae
•Listeria monocytogenes
•Bacillus cereus
•Yersinia enterocolitica
•Campylobacter jejuni
•Plesiomonas shigelloides