WEST BENGAL UNIVERSITY OF
ANIMAL AND FISHERY SCIENCES
FPT - 225
SUBMITTED BY : La Tshering Lepcha
ROLL NO : F/2020/21
SUBMITTED TO : Dr. S. Nath
2
CONTENTS
Practical 1: Organoleptic assessment of
fish freshness and quality of different
fishery
products ..............................................................
.................................................................................
............... Practical 2: Estimation of
pH ..........................................................................
.............................................
Practical 3: Estimation of
TVBN......................................................................
............................................
Practical 4: Estimation of
PV ..........................................................................
.............................................
3
Practical 5: Estimation of free fatty acid
(FFA) .....................................................................
................
Practical 6: Swab
testing ..................................................................
.............................................................
Practical 7: Aerobic Plate Count of
Bacteria ...............................................................
........................... Practical 8: Assessment of
sanitation in fish processing
plant .......................................................
4
PRACTICAL1: ORGANOLEPTIC
ASSESSMENT OF FISH FRESHNESS AND
QUALITY OF DIFFERENT FISHERY
PRODUCTS
1. INTRODUCTION
The quality of fish and fishery products has
become a major concern in fish industry all
over the world (Huss et al., 2003). Fish,
being one of the exceptionally perishable
foods and as a result of globalization of food
trade fish products tend to be more
susceptible to rejection due to poor quality
especially if the initial raw materials are of
poor quality despite the technological
developments in fish production (FAO,
2009). Seafood health hazards have been
5
outlined in several guides in the literature
(FDA, 2001) and can be classified as
(i) biological hazards :(biogenic amines - in
some literature is classified under chemical
hazards or bio toxins, parasites, pathogenic
bacteria, viruses, bio toxins and allergens).
(ii) chemical hazards: (chloramphenicol and
other antibiotic residues for farmed fish,
fish originated from contaminated waters
such as heavy metals, dioxins, chemical
contaminants originated from processing
areas, chemicals formed by fish processing
such as nitrosamines and polycyclic
aromatic hydrocarbons and
(iii) physical hazards :such as bones, plastic,
glass and metals (Huss et al., 2003)
Preservation techniques are needed to
prevent fish spoilage and lengthen shelf life.
They are designed to inhibit the activity of
spoilage bacteria and the metabolic changes
that result in the loss of fish quality.
6
Traditionally processed fish products (TFPs)
are reported to carry high potential risk for
human health for halophilic pathogenic
bacteria, histamine and parasites (Hansen,
2008)
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2. ORGANOLEPTIC METHODS
Highly trained personnel should be
appointed for organoleptic assessment of
quality of fish. Visible signs of deterioration
by
- Sight
- Smell
- Touch
- Taste
3. SENSORY ASSESSMENT
OF FRESH FISH General
appearance:
1) Bright eyes of bony fish.
2) Shiny and brilliant skin.
3) Fishy smell.
4) Bright red colour of gills.
5) Firmness of the fish.
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Quality assessment scheme used to identify
the quality index demerit score (Larsen et
al.1992)
Quality Character Score
parameter (ice/seawater)
0 Bright, shining
Skin
1 Bright
2 Dull
0 None
Bloodspot
General on gill 1 Small, 10-30%
appearanc cover 2 Big, 30-50%
e 3 Very big, 50-100%
0 Stiff, in rigor
Stiffness mortis
1 Elastic
2 Firm
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3 Soft
0 Firm
Belly
1 Soft
2 Belly burst
0 Fresh,
Smell seaweed/metallic
1 Neutral
2 Musty/sour
3 Stale meat/rancid
0 Clear
Clarity
Eyes 1 Cloudy
0 Normal
Shape
1 Plain
2 Sunken
0 Characteristic, red
Colour
Gills 1 Faded, discoloured
0 Fresh,
Smell seaweed/metallic
1 Neutral
2 Sweaty/slightly
rancid
10
3Sour stink/stale,
rancid
Sum of (min. 0 and max. 20)
scores
4. SENSORY ASSESSMENT OF FISH AND
FISHERY PRODUCTS
Sensory evaluation of food is defined as the
scientific means of quantifying and
interpreting the variations in food
characteristics (odour, taste, tactile,
appearance) by using human senses of sight,
smell, taste, touch and hearing. Most sensory
characteristics can only be measured
meaningfully by humans. However,
advances are being made in the development
of instruments that can measure individual
quality changes.
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5. PRINCIPLE
In sensory analysis appearance, odour, flavour
and texture are evaluated using the human
senses. Scientifically, the process can be
divided into three steps. Detection of a
stimulus by the human sense organs;
evaluation and interpretation by a mental
process; and then the response of the assessor
to the stimuli. Variations among individuals in
the response of the same level of stimuli can
vary and can contribute to a non-conclusive
answer of the test. People can, for instance,
differ widely in their response to colour
(colour blindness) and also in their sensitivity
to chemical stimuli. Some people cannot taste
rancid flavour and some have a very low
response to cold- storage flavour. It is very
important to be aware of these differences
when selecting and training judges for sensory
analysis. Interpretation of the stimulus and
response must be trained very carefully in
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order to receive objective responses which
describe features of the fish being evaluated. It
is very easy to give an objective answer to the
question: is the fish in rigor (completely stiff),
but more training is needed if the assessor has
to decide whether the fish is post or pre-rigor.
Subjective assessment, where the response is
based on the assessor's preference for a
product, can be applied in the fields like
market research and product development
where the reaction of the consumer is needed.
Assessment in quality control must be
objective.
6. PROCEDURE
Sensory evaluation of fillets was performed
during storage by a sensory panel composed
of 15 experienced members. They were
required to evaluate the raw fillets based on
the colour, odour, texture and overall
acceptability using a 7-points hedonic scale: 1
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= dislike very much, 2 = dislike moderately, 3
= dislike slightly, 4 = neither like nor dislike,
5 = like slightly, 6 = like moderately, and 7 =
like very much (Siah and Tahir, 2011).
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PRACTICAL 2: ESTIMATION OF pH
1. PRINCIPLE
The hydrogen ion concentration in the fish
samples is is measured with the pH
electrodes/calomel electrode system. The pH
of the muscle can be measured directly using
a combination surface electrode or spear
electrode. Alternatively, the pH of a
homogenate of fish muscle in water can be
measured. This value will differ from the
point measurement.
2. EQUIPMENT
1. pH meter.
2. Combination pH/reference electrode or
separate system.
3. Homogeniser
3. PROCEDURE
1. Homogenise 10g of fish muscle with
20ml of distilled water. As a general rule
the ratio of water to fish should be 2:1.
2. Measure pH of homogenate and correct
to zero dilution according to the
following formula
pH(0) = pH(D) – 0.068 D0.5
where pH(0) = pH at zero dilution
where pH(D) = pH determined on
the homogenate
vol.of water
D = dilution ration
added to
the sample
in ml wt. of
sample in
g
1
1
PRACTICAL 3: ESTIMATION OF TVBN
1. INTRODUCTION
Chemical methods of food evaluation are
normally used to indirectly predict the level
of a sensory attribute, which allows for
immediate determination of freshness. With
regard to evaluation of fish quality using
chemical methods, the total volatile basic
amines constitute to the commonly
measured chemical indicators. Total volatile
base is a general phrase used to include
volatile amines such as, tri methylamine,
ammonia produced by spoilage bacteria; di
methylamine and produced by autolytic
enzymes during frozen fish storage. The
concentration of these chemicals in fish
tissues can be determined by steam
distillation method. Conversely the
measurement of the amount of hypoxanthine
in fish is one of the chemical methods of
2
determining fish freshness. Hypoxanthine is
one of the products of nucleotides
degradation mediated by bacterial activity
(Proteus bacterium) is known to be
responsible for bitter, off flavours of spoilt
fish. Freshness can be determined by
calculating the ratio of inosine and
hypoxanthine to the sum of ATP and all the
other products of ATP degradation
multiplied by 100 (Haard, 2002).
Total volatile basic nitrogen is one of the
most common index of quality adopted
universally for deciding the state of
freshness of fish. It is a general term which
measures the content of trimethylamine,
dimethylamine, ammonia and other basic
nitrogenous compounds associated with the
spoilage of fish. Whereas, ammonia
comprises a major proportion of the TVBN
value. Ammonia is formed by the bacterial
deamination of proteins, peptides and amino
3
acids. It is also produced due to autolytic
breakdown of adenosine monophosphate.
2. PRINCIPLE
Volatile bases are first separated by
precipitating the proteins using the
trichloroacetic acid (TCA) solution. The
filtered solution (TCA extract), which
contains the volatile substances is treated
with strong alkali (K₂CO₃). the liberated
ammonia is trapped by the standard H₂SO₄
solution and the quantity absorbed is
determined by titration of the remaining acid
with the standard NaOH.
3. MATERIALS AND REAGENTS
1. Conway’s micro-diffusion unit
2. Mortar and pestal
3. Paraffin wax and Vaseline in1:2 ratio
4
4. H₂SO₄ : 0.02 N
5. Potassium carbonate solution : saturated
solution is made by dissolving 50 g K₂CO₃
in 100 ml distilled water by heating.
6. Toshiro’s indicator
7. NaOH :0.02 N
8. Trichloroacetic acid (TCA) : 10% solution
4. PROCEDURE
1. Weigh 10 g fish muscle and take in a
mortar
2. Add 20 ml of 10% TCA to it and grind
well with pestle.
3. Filter the content through course filter
paper in 100 ml volumetric flask.
4. Repeat the extraction process twice or trice
to make up the volume to 100 ml (TCA
extract).
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5. TVBN ESTIMATION
1. Wash and dry the Conway cups and lids.
2. Prepare a mixture of melted paraffin wax
and Vaseline in the ratio of 1:2 and cool.
3. Apply this on the rims of cups.
4. Add 1 ml of 0.02 (N) H₂SO₄ into the inner
chamber of each cup.
5. Place lid over the Conway cup covering
part of outer chamber and complete inner
chamber.
6. Add 1ml of TCA extract in to the outer
chamber followed by 1ml of saturated
K₂CO₃ solution.
7. Close the unit with lid and mix the
contents by gently rotating the unit.
(Note : step 6 and 7 should be done as fast
as possible, since ammonia is highly
volatile)
6
8. Leave the unit over night for reaction (it
can be kept inside an incubator at 37 °C
for 2 h.
9. Titrate the excess acid, left in inner
chamber, against 0.02(N) NaOH using a
drop of Toshiro's indicator.
10. Do a blank simultaneously with 10% TCA
solution instead of TCA extract.
[14 × N × (X − Y) × 50 × 100]
6. CALCULATION
TVBN (mg %) =
/W
Wher
e,
N = Normality of H2SO4
X = ml of standard NaOH required for
titration of samples
Y = ml of standard NaOH required for blank
7
W = Weight of the sample taken
8
PRACTICAL 4: ESTIMATION OF PV
1. PRINCIPLE
The sample is treated in solution with a
mixture of acetic acid and a suitable
organic solvent and then with a solution of
potassium iodide. The liberated iodine is
titrated with a standard solution of sodium
thiosulfate.
Peroxides and similar products which
oxidize potassium iodide under the
conditions of the test will contribute to the
peroxide value. Variations in procedure
may affect the results. Peroxide values are
expressed either in milliequivalents of
peroxide/kg or millimoles of peroxide/l.
2. APPARATUS
• Balance, ± 1 mg
• Burette
• Iodine flask, Erlenmeyer flask, 250 ml
with glass stopper
• Stop watch
3. REAGENTS
Chloroform, Cyclohexane, Formic
acid conc.,Glacial acetic acid,
Isooctane Methanol, Starch Sodium
thiosulfate solution, 0.01 mol/l (0.01 N) or
0.1 mol/l (0.1
N) Thyodene Potassium iodide, Water,
dist.
4. PROCEDURE
The sample has to be protected from the air,
stored in a cool place and should not be
opened before the determination is
commenced. Other tests have to be carried
out afterwards. Solid fats may not be melted
before the determination. A sample of fat is
taken from the centre of the sample, and
attention must be paid to the fact that no
sample is taken from the surface. The
sample is transferred into an Erlenmeyer
flask and closed immediately with a glass
stopper. Use 0.1 mol/l sodium thiosulfate
solution when the expected peroxide values
are over 20 mol/l. Use 0.01 mol/l sodium
1
thiosulfate solution when the expected
peroxide values are less than 20 mol/l.
5. CALCULATION
1000(V − X)N
Peroxide value (meq O2 per
1000gm fat) =
W
Where, V= vol. of sodium
thiosulphate used for sample
X= vol. of sodium thiosulphate used
for blank
N= normality of sodium
thiosulphate
W= weight of sample
2
PRACTICAL 5: ESTIMATION OF FREE
FATTY ACID
1. PRINCIPLE
The amount of free fatty acids can be
determined volumetrically by titrating
the sample with potassium hydroxide.
The FFA value of fats and oils is
expressed as the number of mgs of
standard potassium hydroxide required
to neutralize the free fatty acids present
in 1g of fat or oil.
2. REAGENTS
A. Neutral alcohol-to be neutralized
with KOH to a faint but permanent
pink colour just before using. B.
Phenolphthalein indicator .
C. 0.1N KOH solution: 5.6g
potassium hydroxide is weighed and
dissolved in distilled water and the
final volume is made to 1L. This
solution is standardized by titrating
it with a known volume of 0.1N
oxalic acid using phenolphthalein as
an indicator till a permanent pink
colour appears.
3. PROCEDURE
A. Take a known weight of fat or oil in a
clean and dry conical flask.
B. Add 25ml of hot neutral alcohol
followed by few drops of
phenolphthalein indicator.
C. Titrate the content by 0.1N KOH with
vigorous shaking to the first permanent
pink colour of the same intensity as that
of neutral alcohol before addition of the
sample.
D. Ensure that the colour must persist at
least 30 sec.
4. CALCULATION
ml of KOH × (N) of KOH × 56.1
FFA (as oleic acid) % =
Weight of
the sample
Acid value = FFA (%) × 1.99
1
PRACTICAL 6: SWAB TESTING
1. INTRODUCTION
The major changes in fish freshness for
instance unattractive change in food
characteristics such as, flavours, odours
and colour are largely due to bacterial
growth and activity. Microbiological
methods are used to estimate bacterial
numbers, in order to determine fish
freshness, hygiene and evaluate the
possible presence of bacteria or organisms
of public health. Microbiological
estimation of bacterial numbers therefore,
in order to serve the purpose of food safety
and shelf life determination, is expected to
relate quantitatively to the characteristics
of the food during storage (Dalgaard,
2002).
The aim of microbiological examinations
of fish products is to evaluate the possible
presence of bacteria or organisms of public
health significance and to give an
impression of the hygienic quality of the
fish including temperature abuse and
hygiene during handling and processing.
Microbiological data will in general not
give any information about eating quality
and freshness. Traditional bacteriological
examinations are laborious, time-
consuming, costly and require skill in
execution and interpretation of the results.
It is recommended that such analyses be
limited in number and extent. Various
rapid microbiological methods have been
developed during the last decade and some
of these automated procedures may be of
use when large numbers of samples are to
be analysed. Swab test is the counting of
total number of aerobic bacteria, yeasts
and moulds on any surface.
2. PROCEDURE
I. A cotton Swab of Johnsons and
Johnsons make is moistened with
normal saline (0.9 % NaCl) and placed
in a suitable test tube or screw cap test
tube. The mouth of the test tube is
closed with a cotton plug and rapped
with aluminium foil.
II. The test tube containing the swab is then
sterilized by autoclaving at 121ºC, 15
psi, pressure for 15 min.
III. Alternatively pre-sterilised cotton swab
of HI- media make can also be used. In
this case, moisten the swab with sterile
normal saline before using the swab.
IV. Wear handgloves and take out the swab
carefully from the test tube and swab 1
the surface to be checked.
V. The area of the swab should be
approximately 10 Sq. cm.
VI. Replace the swab immediately in the
test tube and close.
VII. Add accurately 10 mL of sterile
phosphate buffer pH 7.2 to the test tube
containing the Swab aseptically.
VIII. Shake well and transfer 1 mL to each of
two sterile petridishes and proceed for
test for Total Bacterial Count and yeast
and mould as per the respective
Standard Testing Procedures.
IX. Also determine the presence of
pathogens if necessary (E. coli,
Salmonella, S.
aureus and Pseudomonas) by
transferring 1 ml to each of the
enrichment
2
PRACTICAL 7: AEROBIC PLATE
COUNT OF BACTERIA
1. INTRODUCTION
Estimation of Aerobic plate count is
intended to give the level of microorganisms
in product and hence indicates the freshness
of the product, the efficacy of a sanitary
practices followed as well as the
effectiveness of the processing technique
employed.
Aerobic Plate Count (APC) is otherwise referred
as Standard Plate Count (SPC) or
Heterotrophic Plate Count (HPC). The APC
can be done by following Spread Plating
Method, Pour Plating Method or Drop Plate
Method. Among all these methods, Spread
Plate Method and Pour Plate Methods are
more common.
2. MATERIALS REQUIRED
I. Sterilised petry dishes
II. Sterilised 1ml pipette/ Micro Pipette tips
III. Sterislised non-selective Medium: For
bacterial enumeration different
nonselective media such as Nutrient Agar,
Plate Count Agar, Iron Agar or Zobell
Marine Agar can be used.
IV. Sterile Diluent: Different diluents such as
Phosphate Buffered Saline (PBS), Normal
saline (0.85% NaCl) or peptone water can
be used. For initial dilution of the sample
during homogenization 225ml or 90 ml of
diluent is sterilized in a conical flask. For
serial dilution, 9ml of diluents are filled in
test tubes and sterilized.
1
V. Homogenizer: For homogenizing the
sample to sample to small pieces and to
bring the sample to suspension either
Omni Mixer, Stomacher assembly or glass
mortar can be used.
VI. Scissors, cotton etc.
VII. Fish Sample.
3. PROCEDURE
Aseptically weigh 25g of the sample and
homogenise in 225ml of sterile diluent i.e.
normal saline. Prepare decimal dilutions as
appropriate, in the same medium by
transferring 1ml of the first dilution to test
tubes containing 9ml of sterile diluent.
4. POUR PLATING METHOD
Pour 1ml of inoculum from each of the
dilutions to sterile petridishes, kept in sets of
duplicates. Pour 15-20ml of Plate Count
Agar or Nutrient Agar medium sterilized and
cooled to 45oC in advance, to each of the
plates. Ensure proper mixing by clockwise
and anticlockwise rotation 5-6 times in a flat
surface and allowed to solidify. Incubate the
plates in inverted condition at 371OC for
24-48 h.
2
