Laboratory Manual On Analytical Methods and Procedures For Fish and Fish Products

LABORATORY MANUAL ON ANALYTICAL METHODS AND
PROCEDURES FOR FISH AND FISH PRODUCTS
Edited by
HIROSHI HASEGAWA
MARINE FISHERIES RESEARCH DEPARTMENT

SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER
SINGAPORE
1987
The Southeast Asian Fisheries Development Center (SEAFDEC) is a technical organisation devoted to the
accelerated development of fisheries in the region. The member countries of SEAFDEC are Japan, Malaysia,
Philippines, Singapore and Thailand. SEAFDEC has three Departments namely, the Aquaculture Department in the
Philippines, the Training Department in Thailand, and the Marine Fisheries Research Department in Singapore.
Southeast Asian Fisheries Development Center

Marine Fisheries Research Department
Postal address: Changi Fisheries Complex
Changi Point, Singapore 1749
Secretariat: Liaison Office

956 Olympia Building
4th floor, Rama IV Road
Bangkok 10500
THAILAND
Copyright © 1987. Marine Fisheries Research Department

Southeast Asian Fisheries Development Center
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any
form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior permission of
the publisher.
Layout:-
MOHAMED BIN SALIM
Typing:-
FLORENCE WONG-LIM KWEE YIN
ISBN 9971-88-158-6
PREFACE
This manual represents a continuing effort by the MFRD to provide useful guides for both
experienced laboratory workers and other technical personnel. Included in the manual are
procedures for determining the physical and chemical properties of fish meat, the analysis of
oils and some additives and microbiological procedures.
The methods described in this manual have been compiled from various sources for use in
MFRD and may not necessarily be the same methods used by other agencies, although
whenever desirable, the officially recognised methods are followed.
In compiling this manual, the contributors have placed emphasis on presenting the
material in this manual as simply and as directly as possible by adopting a step by step
approach for each method discussed. Constructive criticisms are very much welcomed, for the
benefit of future editions. The physical layout of this manual also allows updating of methods
easily.
It is hoped that this publication will be a useful reference tool for those involved in laboratory
work, especially for those working in the fields of fish and fish products analysis.
CONTENTS
PART A. DETERMINATION OF PHYSICAL PROPERTIES OF MEAT
A- 1 Determination of moisture
NG M UI CHNG
A-2 Determination of ash

NG MUI CHNG
A -3 Measurement of pH
LIM PANG YONG
A -4 Measurement of free and expressible drips

NG CHER SIANG
A -5 Fish protein extractibility and its determination

LIM PANG YONG
A -6 Viscosity of fish meat sol

LIM PANG YONG
A- 7 Quality assessment of fish jelly products and raw materials used for
production of fish jelly products
N G MUI CHNG
PART B. DETERMINATION OF CHEMICAL PROPERTIES OF MEAT
B-1 Protein determination by Kjeldahl Method

LIM PANG YONG
B-2 Protein determination by Biuret Method (Modified by Umemoto)

LIM PANG YONG
B-3 Determination of trimethylamine oxide (TMAO-N), trimethylamine (TMA-N),

total volatile basic nitrogen (TVB-N) by Conway’s Method
NG CHER SIANG
B-4 Determination of DMA-N by Dyer’s Colorimetric Method using copper

dithiocarbamate
NG CHER SIANG
B-5 Determination of formaldehyde in fish meat using Nash’s Reagent

NG CHER SIANG
B -6 Determination of K-value
NG CHER SIANG
B-7 Freshness testing paper

NG CHER SIANG
PART C. ANALYSIS OF OILS
C-1 Significance of analysis of lipids

LOW LAI KIM & NG CHER SIANG
C-2 Extraction of lipids (modified Folch’s Method)

C-3 Determination of total lipid content

C -4 Determination of phospholipid content

C -5 Determination of acid value

C-6 Determination of saponification value

C -7 Determination of peroxide value

C -8 Determination of thiobarbituric acid (TBA) number

C -9 Determination of methyl esters of fatty acids by gas chromatographic

method
C-10 Determination of the degree of lipid oxidation by chromatography

C-11 Preparation of methyl esters by Boron Triflouride Method

PART D. ANALYSIS OF ADDITIVES
D-1 Detection of polyphosphates

NG MUI CHNG
D-2 Determination of monosodium L-glutamate (MSG) content in fish jelly

products
NG MUI CHNG
D-3 Determination of sugar (sucrose) by Somogyi’s Method

NG MUI CHNG
D-4 Determination of starch

NG MUI CHNG
D-5 Determination of salt

NG MUI CHNG
D-6 Semi-quantitative analysis of boric acid and borates in meat and meat
products
NG MUI CHNG
PART E. MICROBIOLOGICAL PROCEDURE
E-1 Handling of food samples

LIM PANG YONG
E-2 Aerobic plate count

LIM PANG YONG
E-3 Coliforms and Escherichia coli

LIM PANG YONG
E-4 Salmonellae and Shigella

LIM PANG YONG
E-5 Staphylococcus aureus

LIM PANG YONG
E-6 Faecal streptococci

LIM PANG YONG
E-7 Vibrio cholera

LIM PANG YONG
E-8 Vibrio parahaemolyticus

LIM PANG YONG
APPENDICES
A Most probable numbers (MPN) per 1 g of sample, using 3 tubes with each of
0.1, 0.01 and 0.001 g portions
B Media preparation methods
C Biochemical tests in diagnostic microbiology
D Preparation methods for reagents

DETERMINATION OF PHYSICAL
PROPERTIES OF MEAT
DETERMINATION OF MOISTURE
NG M .C .
INTRODUCTION
There are various methods to determine the moisture content. The determination depends
on the following criteria:-
a) the form in which water is present

b) nature of product analysed
c) rapidity of determination
d) accuracy desired
e) availability and cost of equipment required
In the case of fish meat, the methods used are oven method, rapid methods by infra-red
balance and microwave moisture checker.
I SAMPLE PREPARATION
Collect meat sample (≤ 100 g) and pass 2-3 times through food mincer, or chop very finely
and mix thoroughly.
II INSTRUMENT
Method 1: oven (30-250°C), aluminium dish with lid.
Method 2: infra-red balance (Kett, model F-1A).
Method 3: microwave moisture checker (Anritsu, model K377C).
III ANALYTICAL PROCEDURE AND CALCULATION

METHOD 1: OVEN METHOD
1. Dry the empty dish and lid in the oven at 105°C for 30 min and transfer to the desiccator to
cool (30 min). Weigh the empty dish and lid to 3 decimal places.
2. Weigh about 5 g of sample from (I) to the dish. Spread the meat with spatula. Replace the
lid and weigh the dish and contents to 3 decimal places.
3. Place the dish with its lid partially covered in the oven. Dry for 16 hrs or overnight at 105°C.
4. After drying, transfer the dish with partially covered lid to the desiccator to cool. Reweigh
the dish and its dried content.
A- 1.1
CALCULATION
W 1 - W2
Moisture (%) = x 100
W1
where W 1 = weight (g) of sample before drying.

W 2 = weight (g) of sample after drying.
METHOD 2: INFRA-RED METHOD

1. Balance the infra-red meter at zero level.
2. Evenly spread accurately 5 g meat sample from (I) onto the dish.
3. Place dish with sample on infra-red meter dish holder and level the balance.
4. Set lamp height to mark 7 and switch on the moisture meter. As moisture content in the
sample decreases, lower the lamp height gradually until mark 5-4.5.
5. Continue to dry until the readout on the scale is constant (30-45 mins).
CALCULATION
(a) Results can be read directly from the balance scale
(b) Calculate similarly the oven method i.e.;
W 1 – W2
Moisture (%) = x 100
W1
where W 1 = weight of sample before drying.

W 2 = weight of sample after drying.
METHOD 3: MICROWAVE METHOD

1. Warm up and stabilise the microwave checker for half an hour before use.
2. Tare the sample dish containing glass fiber filter and Teflon ring to zero.
3. Evenly spread about 5 g meat sample on the sample dish and cover with filter paper held
in place with Teflon ring.
4. Close the oven door. The weight of sample (g) is displayed on readout.
5. Set the required time at full power, 600w and at variable power, 300w (see below table).
6. Press the start switch to activate the drying.
7. At the end of drying, a buzzer sounds and the moisture content (%) is displayed directly.
8. Press the readout button to obtain the dried weight.
9. Repeat additional 30 sec at 300w until dried weight is constant.
A—1.2
SUITABLE TIME AND HEATING CONDITIONS FOR FISH MEAT SAMPLE
Power
Sample 600w 300w
Minced meat 120sec 60sec
Leached meat 300sec 30sec
Surimi 120sec 90sec
CALCULATION
The microwave method is calibrated to give direct readout in % moisture.
A- 1.3
DETERMINATION OF ASH
NG M. C.
INTRODUCTION
The principle of ashing is to burn off the organic matter and to determine the inorganic
matter remained. Heating is carried out in two stages:- firstly to remove the water present and
to char the sample thoroughly; and finally ashing at 550°C in a muffle furnace.
This method is applicable to all food materials.
Randomly collect meat sample (≤ 100 g) and pass through a manual mincer twice or chop
very finely and mix thoroughly.
Place minced meat in small plastic bag.
II INSTRUMENT AND APPARATUS

Muffle furnace, temperature (0-1200)°C
Crucibles and lids
Thong
Thick gloves
III ANALYTICAL PROCEDURE

1. The crucible and lid is first placed in the furnace at 550°C overnight to ensure that
impurities on the surface of crucible is burnt off. Cool the crucible in the desiccator (30
mins).
2. Weigh the crucible and lid to 3 decimal places.
3. Weigh about 5g meat sample from (I) into the crucible. Heat over low bunsen flame with lid
half covered. When fumes are no longer produced, place crucible and lid in furnace.
4. Heat at 550°C overnight. During heating, do not cover with the lid. Place the lid on after
complete heating to prevent loss of fluffy ash. Cool down in the desiccator.
5. Weigh the ash with crucible and lid to 3 decimal places.
6. Ash must be white or light grey. If not, return the crucible and lid to the furnace for further
ashing.
IV CALCULATION
Wt of ash
Ash Content (%) = x 100
Wt of sample
REFERENCE
Official methods of analysis of the Association of Official Analytical Chemists 13th Ed., 1980:
289, 508. See 18.025, 31.012.
A-2.1
MEASUREMENT OF pH
LIM P. Y.
INTRODUCTION
Pre-rigor fish flesh is semi-translucent, glossy and dry in appearance and no moisture can
be expressed from it. The flesh is nearly neutral, that is its pH, the degree of acidity or alkalinity
is near 7.0. After rigor has resolved, the flesh is wetter in appearance, moisture can be
expressed much more easily from it and pH is more acid. The lowering of pH is due to the
breakdown of glycogen to lactic acid. Depending upon species, the pH immediately after rigor
has resolved is usually 6.4 to 6.8. The pH increases again with increased growth of spoilage
bacteria.
The pH of the environment affect bacterial growth. Most bacteria especially spoilage
bacteria grow well between pH 6 and pH 8 with progressively less growth at extremes of pH.
On the other hand, the pH of other animal meats is between 5.3-6.0, the bacteria grow less
readily. This is one reason why fish spoil more quickly than meat. However, the measurement of
pH is an indicator of fish freshness.
I SAMPLING AND SAMPLE PREPARATION

Take a representative sample from the experimental material or product lot, avoid taking
the red meat portion and store in polyethylene bag prior to preparation for analysis. The
sample should be kept in refrigerator or in ice to maintain its integrity.
Homogenise the sample with a mechanical/electrical mincer or chop the sample with knife
until homogeneous.
Transfer the homogenised sample into a polyethylene bag and store in refrigerator until
required. Ensure that the prepared sample is still homogeneous prior to weighing.
In case of fresh fish meat, the pH of fish homogenate should be determined at once.
II APPARATUS
Round bottom flask
Heating mantle
Mortar & pestle
Tissue homogeniser or grinder with speed control
Beakers, 100 ml
pH meter
III REAGENTS
a) CO2 free distilled water
Boil the distilled water with a round bottom flask. Cool the distilled water prior to use. Cap
the flask to avoid contact with atmospheric air.
b) Treated sand
Sieve the sea sand and wash the resulting fine sand 3 times with distilled water.
Boil the washed sand for 15 mins. in a 1N NaOH solution. Allow to cool.
A-3.1
Decant away the NaOH solution and wash the sand 3 times with distilled water or until it is
free of the alkali.
Boil the sand for 15 mins. in a 1N HCl solution. Allow to cool.
Decant away the HCl solution, and wash the sand 3 times with distilled water or until it is
free of the acid.
Place the treated sand in an oven set at 105°C overnight to dry.
IV PROCEDURES
1. Sample prepared with mortar and pestle
Weigh accurately 2.0 g of sample and place into a mortar.
Add approximately 2.0 g of treated sand to the mortar and grind until the sample is
homogenised.
Add 10 ml of CO2 free distilled water to the homogenate and grind again.
Remove the well ground homogenised sample into beaker and read the pH.
2. Sample prepared with tissue homogeniser/grinder

Weigh accurately 5.0g of sample and place into the beaker.
Add 45 ml of CO2 free distilled water to the sample and homogenise for 30 seconds.
Read pH of sample.
A-3.2
MEASUREMENT OF FREE AND EXPRESSIBLE DRIPS
NG C. S.
INTRODUCTION
When animal tissues (eg. muscle) are frozen, a certain degree of damage occurs. In
muscle tissue, this is reflected in an increased amount of free drips and expressible drips. Free
drips is the fluid that exudes from the muscle (or thawed muscle) on standing. Expressible drip
is the fluid lost from the meat on application of pressure. No standard method has been
established for drips measurements, and the amount of drips measured is a relative value. On
reporting drip values, it is therefore important to state the physical parameters involved.
I APPARATUS
2 cm Ø cork borer
Petri dishes
Filter paper (Whatman No. 1 , Ø 7 cm)
Screw press
Stop watch
II PROCEDURE
1. A 2cm Ø cylinder of fish muscle is made using the cork borer. Trim the muscle block of
the skin and cut the height to 0.5 cm.
2. Weigh the muscle sample (X g) and place it on 2 pieces of filter paper. Place sample in a
petri dish with cover. Keep in refrigerator (4°C) for 2 hr.
3. Take the sample out and weigh (Y g).
4. Place the muscle sample between 2-filter paper on top and 3-filter paper below. Place the
whole in the press.
5. Slowly increase the pressure to 10 kg/cm2 within 30 sec.
6. Maintain at 10 kg/cm2 constant pressure for 2 min, then remove the sample, and weigh
the pressed sample (Z g).
III CALCULATION
(X – Y)
Free drip, % = x 100
X
(X – Z)
Expressible drip, % = x 100
X
A–4.1
IV PRECAUTIONS
a) The cut sample must be kept frozen until ready for weighing.
b) Maintain a constant size of sample. Sample size approx. 0.5 cm in thickness and 2.0 cm in
diameter.
c) Take muscle samples from a constant area of the fish.
d) Weigh the frozen sample quickly to prevent atmospheric moisture from condensing on the
sample.
A- 4.2
FISH PROTEIN EXTRACTIBILITY & ITS DETERMINATION
LIM P.Y.
INTRODUCTION
Fish proteins gradually become denatured in cold storage. The rate of denaturation
depends largely upon storage temperature.
Badly frozen stored fish are easily recognizable. The appearance of the thawed product,
instead of being glossy and translucent, is dull and opaque and the texture, no longer firm and
elastic, becomes soft and spongy. The cooked flesh loses its succulence and becomes dry,
fibrous and tasteless.
The main proteins of fish flesh are called myosin and actin. They are responsible for the
mechanism of contraction and relaxation of muscles and are called myofibrillar proteins.
Muscle also contains many other proteins, the sarcoplasmic protein which are soluble in tissue
fluid and in any salt solution. During freezing and cold storage, the proteins are affected,
especially the myofibrillar protein, resulted in the textural changes of flesh.
The myofibrillar protein extractibility, therefore is used as a quality index for the
assessment of frozen fish. It is expressed as follows:-
MPN – SPN
Extractibility (%) = x 100 (A)
TN – NPN
where: MPN = myofibrillar protein-nitrogen (N mg/100 g sample)

SPN = sarcoplasmic protein-nitrogen (N mg/100 g sample)
TN = total nitrogen (N mg/100 g sample)
NPN = non proteinous nitrogen (N mg/100 g sample)
The protein extractibility is applicable to fish and its product in general and can be used as
an indicator of the degree of protein denaturation for demersal and pelagic species during cold
storage.

Take a representative sample 22 g or more from the product. Place the sample in
polyethylene bag and store in refrigerator or in ice so as to maintain sample integrity before
preparation for analysis.
The dark meat, if any, should be removed prior to homogenisation of fish flesh.
Comminute the sample until homogeneous and place the homogenate in a polyethylene
bag. Store the sample in a refrigerator or in ice until required. Ensure that the prepared
sample is still homogeneous prior to weighing.
A-5.1
II APPARATUS
Chopper or mincer
Analytical balance, decimal to 0.1 mg
Spatula
Bottom-drive homogeniser (Nihon Seiki SN-03) or equivalent
Refrigerated centrifuge, capable of centrifuging at 12,500 g
Beakers, 100 and 250 ml
Bulb pipettes, 10, 20 & 40 ml
Glass funnels 0 60 mm
Whatman filter paper, No. 41
III REAGENTS
a) Phosphate buffer solution
0.03 M potassium di-hydrogen phosphate, 1 litre.
0.03 M di-sodium hydrogen phosphate, 1 litre.
Mix the above solutions into a 2 litre beaker.
Adjust the pH to 6.85 using these solutions.
Store in refrigerator.
b) 0.1 M potassium chloride solution
Weigh KCl required accurately, use distilled water as solvent.
c) 0.5M potassium chloride buffered solution

Accurately weigh KCl required. Dissolve the weighed KCI in the required phosphate buffer
solution.
d) Trichloroacetic acid solution (25%, w/v)

Dissolve 25 g TCA in 75 ml distilled water.
IV PROCEDURE OF PROTEIN EXTRACTIBILITY

1. Total nitrogen. Accurately weigh a duplicate of 1 g the homogeneous fish sample for
protein digestion (refer to protein determination by Kjeldahl method B-1.)
2. Sarcoplasmic protein nitrogen. Accurately weigh 10 g of the homogeneous fish sample.

Blend the sample with 200 ml of 0.1 M KCI solution with the homogeniser for 4 mins
(speed set at scale 2). Leave to stand in iced water for 2 hrs. Centrifuge the blended
sample at 12,500 g or 9,000 rpm at 5°C for 20 mins. Pipette 20 ml supernatant
(sarcoplasmic protein fraction) for digestion (refer to Kjeldahl method).
3. Myofibrillar protein nitrogen. Accurately weigh 10 g of the homogeneous fish sample.

Blend sample with 200 ml 0.5 M KCl phosphate buffered solution with the homogeniser for
4 mins (speed set at scale 2). Leave to stand in iced water for 2 hrs. Centrifuge the blended
sample at 12,500 g or 9,000 rpm at 5°C for 20 mins. Pipette 20 ml supernatant (myofibrillar
protein aliquot) for digestion (refer to Kjeldahl method).
4. Non proteinous nitrogen. Pipette 40 mi of sarcoplasmic protein aliquot (see Section IV 2)

into 100 ml beaker. Add 10 ml 25% TCA and leave to stand in iced water for 30 mins with
occasional swirling. Filter the content of the beaker with Whatman No. 41 ashless filter
paper. Pipette 40 ml of filtrate for digestion (refer to Kjeldahl method).
A-5.2
V CALCULATIONS
Ws in formula (1) in the protein determination by Kjeldahl method (B-1 Section IV) has to be
replaced by the meat weight (g) in each of the protein aliquot used as follows:-
1) For myofibrillar protein nitrogen (MPN)
20
WMPN = W1 x
W1 + 200
where W1 = weight of fish flesh (g) used for myofibrillar protein extraction
20 is the volume (ml) of sarcoplasmic protein aliquot used for digestion
200 is the volume (ml) of 0.5 M KCI — buffered solution used for the extraction
of myofibrillar protein
2) For sarcoplasmic protein nitrogen (SPN)
20
WSPN = W2 x
W2 + 200
where W2 = weight of fish flesh (g) used for sarcoplasmic protein extraction
20 is the volume (ml) of sarcoplasmic protein aliquot used for digestion
200 is the volume (ml) of 0.1 M KCl solution used for the extraction of
sarcoplasmic protein
3) For non-proteinous nitrogen (NPN)
40b W2
WNPN = 40a x X
50 200 + W2
where 40a is the volume (ml) of supernatant of sarcoplasmic protein aliquot used for
non-proteinous nitrogen
40b is the volume (ml) of filtrate used for digestion taken from the sarcoplasmic
protein aliquot after preciptiated by 25% TCA
50 is the total volume (ml) of sarcoplasmic protein aliquot after addition of 10
ml 25% TCA
W2 = weight of fish flesh (g) used for sarcoplasmic protein extraction

200 is the volume (ml) of 0.1 M KCl solution used for the extraction of
sarcoplasmic protein
Use the above equivalent meat weights and the formula (1) in protein determination by
Kjeldahl method (B-1 Section IV) for calculation of respective protein nitrogen aliquot, express
in mgN/100 g and calculate the extractibility by formula (A).
N.B. The pH plays an important role in the extraction of fish protein. The optimum pH is
between 6.5-7.0. Adjustment of the pH of the buffer solution is important and can be
achieved by using di-sodium hydrogen phosphate, potassium di-hydrogen phosphate or
sodium carbonate etc.
A-5.3
VISCOSITY OF FISH MEAT SOL
LIM P.Y.
INTRODUCTION
Viscosity is the measure of fluid friction. It may be considered as the internal friction
resulting when a layer of fluid is made to move in relationship to another layer. A highly viscous
material is one possessing a great deal of internal friction — it will not pour or spread as easily
as a material of lesser viscosity.
This procedure can be used as a rapid method to assess the gel forming ability of the fish
meat, fish mince, leached meat and surimi etc. Generally, fish flesh with meal sol of a minimum
viscosity between 300-400 centipoises can be used to process good quality fish jelly products
(e.g. fishballs or fishcakes).
Practically all fluids will become less viscous as their temperature increases, and thicker as
they cool. The relationship between viscosity and temperature is exponential in nature; that is, a
small temperature change can cause a large viscosity change. The temperature of the material
MUST be stated along with its viscosity. Not to do so nullifies the meaning of the resulting
viscosity value.
The relationship between viscosity and meat concentration is in the form of a power curve.
As such, it is important that the meat concentration be constant for comparative studies.
There is a maximum speed at which layers of fluid can move with laminar flow; that is, with
no transfer of matter between the layers. Turbulence results beyond this maximum speed, and
to maintain this turbulent flow, a larger energy input is necessary. This is reflected by an
apparently higher internal friction, and the indicated viscosity will be higher than it should be.
The Tokyo Keiki Rotary Viscometer functions at a constant speed of 20 rpm.

Take a representative sample minimum of 70 g from the product. Place the sample in
polyethylene bag and store in refrigerator or in ice so as to maintain sample integrity, in
preparation for analysis.
Comminute the sample with a chopper or mechanical mincer until homogeneous and
place the homogenate in a polyethylene bag. Store the sample in the refrigerator or in ice
until required. Ensure that the prepared sample is still homogeneous prior to weighing.
II APPARATUS
Bottom-drive homogeniser (Nihon Seiki SN-03) or equivalent Rotary Viscometer Type C,
CVR-20B, Tokyo Keiki, with 2 spindles (one with a factor of 5 for less viscous fluids and the
other with a factor of 20 for viscous fluids)
Beaker, 1000 ml
Chopper or mechanical mincer
Spatula
A-6.1
III REAGENTS
a) Sodium chloride, extra pure.
b) Di-potassium hydrogen orthophosphate (KH2PO4), cryst. extra pure.
c) Potassium dihydrogen orthophosphate (KH2PO4), cryst. extra pure.
d) Extraction solution: Dissolve 189 g NaCl, 33.5 g K2HPO4 and 8.74 g KH2PO4 in 1000 ml
distilled water. Transfer the solution in reagent bottle and store in refrigerator.
IV PROCEDURES
1. Weigh ca 70 g meat sample into the cylinder of SN type homogeniser.
2. Add 500 g chilled distilled water (ca 10°C) in the cylinder of homogeniser.
3. Completely remove the air bubble in the meat sample with slow speed of homogeniser.
4. Add 100 ml extraction solution and homogenise for 3 mins with speed dial at 3-4.
5. Transfer the meat sol to 1 litre beaker and keep in ice water (below 5°C) for 20 mins.
6. Measure viscosity of the sol with Type C viscometer with the guard, mesh and selected
spindle (temperature of the meat sol should be about 7-10°C).
7. Read the viscometer when the pointer stabilised and note the temperature of the meat sol.
V CALCULATION
Multiply the viscometer reading by 5 if the large spindle is used or by 20 if the smaller
spindle is used and express the viscosity of the meat sol in centipoises.
A-6.2
QUALITY ASSESSMENT OF FISH JELLY PRODUCTS AND
RAW MATERIAL USED FOR PRODUCTION OF FISH JELLY
PRODUCTS
NG M. C.
INTRODUCTION
The quality offish jelly products is assessed by measuring the gel strength objectively and
organoleptically by folding and teeth-cutting tests. This quality depends on the following
factors:-
1. Fish species
2. Condition of fish
3. Processing method and control
4. Moisture content of final product
The quality assessment would be useful for raw material suppliers and its users (eg fishball
processors) to know the quality of the raw material used for the production of fish jelly products;
and to assess the quality of the final products.
I INSTRUMENT/APPARATUS AND MATERIAL

Sausage casing (Ø ca 2.5 cm)
Fudoh Rheometer (Model NRM-2002J).
Knife
Cutting board
Trays
Stainless steel moulds — i) Ø 2.5 cm, 2.4 cm thick for gel strength measurement
ii) Ø 2.5 cm, 5 mm length for organoleptic assessment
II SAMPLE AND TEST PIECE PREPARATION

For raw material
1. Randomly collect 300 g raw material sample (eg minced meat, leached meat, surimi).
2. Put raw material sample into the mortar grinder.
3. The raw material sample is ground for 25 min as follows:-

i) grind the sample for 5 min to break up the muscle fibres.
ii) add 1.5% salt based on the weight of fish meat and grind for 5 min.
iii) add another 1.5% salt and grind for another 5 min.
iv) add water 30% gradually to the ground meat sample and continue to grind for 10min
with constant mixing.
A- 7.1
4. Fill the ground fish paste into sausage casing taking care not to include air bubbles. This is
done by pressing the meat paste onto a board before filling into the casing.
5. Set the sausage-like sample in water bath at 40°C for 20 min followed by heating at 90°C
for 20 min.
6. After heating, cool the sample in iced water immediately to prevent further heating.
7. Immerse the sausage-like sample in running water till sample is at room temperature
before measurement.
8. Cut the sample for gel strength measurement into 2.4 cm length, Ø 2.5 cm with the
stainless steel moulds and place on a tray. Five test pieces will be measured for each
sample. Slice 5 test pieces of 5 mm thickness. Ø 2.5 cm for the organoleptic assessment.
For fish jelly products

1. Fish jelly products eg fishballs, fishcakes, must have a height and thickness of ca 2.0-2.4
cm.
2. These products will be trimmed into standard size of 2.0-2.4 cm by 2.0-2.4 cm.
3. Prepare 5 test pieces for gel strength measurement.
4. Slice 5 test pieces of 5 mm thickness for organoleptic assessment.
Ill MEASUREMENT AND ASSESSMENT

A. GEL STRENGTH MEASUREMENT BY FUDOH RHEOMETER
1. Set the following parameters on the Fudoh rheometer and chart recorder:-
Rheometer Chart Recorder Factor

Speed (cm/min) 6 12 (x ½)
Sensitivity (volts) 1 ½ (x ½)
2. Place a test piece on the sample holder and ‘ON’ the Fudoh rheometer and chart recorder
simultaneously.
3. When test piece is broken as indicated in the recorder chart, ‘OFF’ the chart recorder and
rheometer.
4. Repeat with all the test pieces to obtain the average results.
A-7.2
CALCULATION
Gel strength = X x Y x F g.cm
1 1 1
where F, factor = X =
2 2 4
Fish jelly products of acceptable grade have a gel-strength of 200-300 g.cm.
B. ORGANOLEPTIC ASSESSMENT BY FOLDING AND TEETH-CUTTING TESTS

Organoleptic tests provide a convenient and quick assessment of the “ springiness” of
final products. Although the tests should be performed by trained personnel, the training is
well worth the effort.
(a) Folding Test

Five slices of 5 mm thickness are taken from the prepared samples. Each is then
folded in half and if there is no tear or breakage, further folded into quarter. The
grading is as follow:-
Condition of test samples when folded Grade

• No breakage in any of five samples when folded in quarter AA
• Slight tear in any one of five samples when folded in quarter A
• Slight tear in any one of five samples when folded in half B
• Breakage (but 2 pieces still connected) when folded in half C
• Breaks completely into 2 pieces when folded in half D
Commercial products of acceptable grade should have a rating of AA.
(b) Teeth-Cutting Test

Samples similar to that for folding test are used to assess the “ springiness” using the
teeth-cutting test. The grading gives subjective assessment of the resistance
experienced by a trained panel when the test piece is bitten between the upper and
lower front teeth.
A-7.3
Score Grade
10 Extremely strong springiness
9 Very strong springiness
8 Strong springiness
7 Quite strong springiness
6 Acceptable springiness
5 Acceptable, slight springiness
4 Weak springiness
3 Quite weak springiness
2 Very weak springiness
1 Mushy texture, no springiness
Local products usually fall in the range of score 5-6.
REFERENCE
Instruction and operational manual, Fudoh rheometer
Fudoh Kogyo Co. Ltd. Available in MFRD laboratory.
A-7.4
DETERMINATION OF CHEMICAL
PROPERTIES OF MEAT
PROTEIN D ETER M IN ATIO N BY KJELDAHL M ETH O D
LIM P.Y.
INTRODUCTION
In the presence of sulphuric acid and catalyst, the nitrogen atom in the nitrogenous
organic compound is converted to ammonium sulphate. The ammonia is then distilled from an
alkaline medium and absorbed in boric acid. The ammonia is then determined by titration with
a standard mineral acid.
Taking protein as an example, it is as follows:-
h 2so 4
Protein N (NH4)2SO4 + CO2 ↑ + H2O
Catalyst
(NH4)2SO4 + NaOH → Na2SO4 + 2NH4OH
NH4OH → NH3 + H2O
3NH3 + H3BO3 → NH4 + BO3
BO3 + 3H + → H 3B O 3
I APPARATUS
Kjeldahl digestion and assembly (“ Tecator” brand)
Kjeldahl digestion tube, 250 ml
Kjeldahl distillation apparatus (“ Tecator” brand)
Conical flask 250 ml
Autom atic burettes 50 ml with 2000 ml reservoir bottle
Magnetic stirrer
II REAGENTS
a) Sulphuric acid (H2SO4), nitrogen free
b) Catalyst
Mix 9 parts of potassium sulphate (K2SO4) anhydrous, nitrogen free with 1 part of copper
sulphate (CuSO4), anhydrous, nitrogen free.
c) NaOH solution (40% w/v)

Dissolve sodium hydroxide (NaOH), technical grade, mini pearls, in distilled water.
d) Boric acid (4% w/v)
e) Anti-bumping granules
f) Ethanol (95% v/v)
g) Standard 0.1N Sulphuric acid

Break ampoule for preparation of standard solution, em pty content into 1 L volumetric
flask and dilute with nitrogen free distilled water until the mark. Cap and invert the
volumetric flask until solution well mix. Transfer the content to automatic burette.
h) Indicator
Mix 100 ml of 0.1 % methyl red (in 95% ethanol) with 200 ml of 0.2% bromocresol green (in
95% ethanol).
B- 1.1
III PROCEDURE
1. Accurately weigh the homogenous fish sample (1 g) or pipette a suitable quantity of
protein fraction solution (20 ml m yofibrillar or sarcoplasmic protein fraction or 40 ml
non-proteinous nitrogen fraction) and place in digestion tube. Add 7 g catalyst, 3 to 5
anti-bum ping granules and 20 ml of cone. H2SO4. Also prepare a tube containing the
above chemicals except fish sample as blank. Cover tube with exhaust manifold and place
tube in the preheated digestor and digest at about 110-130°C for 15 mins (ignore this
process if non liquid sample is to be digested). Turn the digestor to digestion temperature
normally around 420°C and digest the sample until the solution is light green (1 hr for fish
sample) and then a further 15 mins. Remove tube and leave to stand until sample is
cooled. Add cautiously 60 ml distilled water.
2. Switch on distillation apparatus and pre-wash for 10 mins. Dispense 25 ml 4% boric acid
into a 250 ml conical flask and place the flask under the condenser, ensuring that the
condenser tip is immersed in the boric acid solution. Connect the digestion tube
containing the sample digest to the distillation apparatus. Dispense 60 ml 40% NaOH
carefully into digested sample. Immediately turn on the steam supply valve to initiate the
distillation. Heat for 4 mins until all ammonia has passed over into the boric acid. Lower the
conical flask ensuring the condenser tip is not immersed in solution and continue heating
for further 1 min. Collect approximately 120 ml distillate. Wash tip of condenser with
distilled water.
Place conical flask containing ammonia distillate on magnetic stirrer. Add 1 ml indicator
and titrate the sample with standard 0.1N sulphuric acid until the solution change from
green to pinkish. Read volume of acid used for titration.
IV CALCULATIONS
Calculate the protein nitrogen (mgN/100 g or 100 ml sample) as follows:-
a) solid/sem i-solid fish sample

(b - a) x 0.1 x 14.00
protein nitrogen = x 100 (1)
Ws
where Ws = weight (g) or volume (ml) of sample
a = volume (ml) of 0.1N H2SO4 used in blank titration
b = volume (ml) of 0.1N H2SO4 used in sample titration
14.00 = atom ic weight of nitrogen
b) Calculation of percentage protein

The above protein nitrogen (mgN/100 g or 100 ml sample) can also be presented as
percentage protein nitrogen fraction and is expressed as follows:-
(b - a) x 0.1 x 14.00 6.25

% protein = x 100 x
W 1000
(b - a) x 0.1 x 14.00
where x 100 is similar to formula (1)
Ws
1000 : the conversion of m gN/100 g to gN /100 g sample
6.25 : the protein-nitrogen conversion factor for fish and its by-products.
B -1.2
P R O T E IN D E T E R M IN A T IO N B Y B IU R E T M E T H O D
( M O D IF IE D B Y U M E M O T O )
L IM P .Y .
IN T R O D U C T IO N
T h is m e th o d is a p p lic a b le to e x tra c te d liq u id fish p ro te in a liq u o ts (See A -5 S e c tio n IV, 2

a n d 3) w ith a p ro te in c o n c e n tra tio n o f b e tw e e n 0.1 to 0 .5 m g N /m l.
T he m e th o d is b a s e d on th e re a c tio n o f C u ++ w ith p e p tid e s in alka lin e s o lu tio n to yie ld a

p u rp le C u ++ — p e p tid e c o m p le x th a t has a p e a k o f a b s o rp tio n at 54 5 nm .
S o m e fis h p ro te in fra c tio n s c o n ta in in te rfe rin g s u b s ta n c e s w h ic h ca u se tu rb id ity to th e

s a m p le s o lu tio n w h e n th e s a m p le is le ft to s ta n d fo r a tta in m e n t o f ch e m ic a l e q u ilib ra tio n (for fu ll
c o lo u r d e v e lo p m e n t). T h e s e s u b s ta n c e s in c lu d e tris — (h yd ro xym e th yl) m e th y la m in e u sed as
b u ffe rin g re a g e n t d u rin g th e e x tra c tio n o f fis h p ro te in a nd s u c ro s e & s o rb ito l u sed as
c ry o p ro te c tiv e re a g e n t in m in c e d fis h fle s h d u rin g fro ze n sto ra g e . O th e r in te rfe rin g c h e m ic a ls
are a m m o n iu m s u lp h a te , m e rc a p to -e th a n o l, T rito n X -1 0 0 e tc. T h e re fo re th is m e th o d is n o t
s u ita b le fo r s a m p le s c o n ta in in g th e a b o v e in te rfe rin g su b s ta n c e s .
I APPARATUS
B u lb p ip e tte , 5 ml
Q u ic k fit te s t tu b e w ith s to p p e r, 25 ml
T e s t tu b e s h a k e r
S p e c tro p h o to m e te r
M a g n e tic s tirre r
B e a k e r 2 5 0 ml
II REAGENTS
a) C o p p e r s u lp h a te p e n ta h y d ra te (C uS O 4-5 H 2O)
b) S o d iu m h y d ro x id e (NaO H)
c) G ly c e rin e
d) R e agen t A.
D isso lve 8 g N aO H in 4 0 m l d is tille d w a te r. A d d th e N aO H s o lu tio n to 30 m l d is tille d w a te r

c o n ta in in g 0.2 g g ly c e rin e . D isso lve 0 .4 g C u S O 4-5 H 2O in 30 m l d is tille d w a te r, a d d th is
s o lu tio n s lo w ly to th e a b o v e m ix tu re s o lu tio n w ith c o n tin u o u s a g ita tio n to p re v e n t
p re c ip ita tio n . T h is s o lu tio n s h o u ld n o t be k e p t in re frig e ra to r fo r m o re th a n 2 m o n th s.
e) R e agen t B.
D isso lve 8 g N aO H in 8 0 m l d is tille d w a te r. W e ig h 0.2 g g ly c e rin e a nd d is s o lv e it in 2 0 ml

d is tille d w a te r. M ix th e s e tw o s o lu tio n s a nd keep in re frig e ra to r and it s h o u ld n o t b e k e p t
fo r m o re th a n 2 m o n th s .
B-2.1
Ill PROCEDURE
A. PR EP AR A TIO N O F C A L IB R A T IO N CU RVE
1. T he B o vin S e ru m A lb u m in o r m y o fib rilla r p ro te in e x tra c t fro m fish can be used as w o rk in g

s o lu tio n fo r th e p re p a ra tio n o f c a lib ra tio n curve.
a) B o vin S e ru m A lb u m in
S to c k s o lu tio n : D isso lve a b o u t 4 0 0 m g a lb u m in in d is tille d w a te r a nd d ilu te to 50 ml

(a b o u t 8 m g /m l)
W o rk in g s o lu tio n : P ip e tte 5 m l o f s to c k s o lu tio n in to 1000 m l v o lu m e tric fla s k a n d d ilu te

w ith d is tille d w a te r (a b o u t 0.4 m g /m l).
b) M y o fib rilla r p ro te in e x tra c t
It is p re fe ra b ly to use th e m y o fib rilla r p ro te in e x tra c t o b ta in e d fro m th e sa m e g ro u p o f fish

fo r th e p re p a ra tio n o f c a lib ra tio n curve.
T he p ro te in e x tra c t has to b e d ig e s te d fo llo w in g K jeld ahl m e th o d a nd th e n itro g e n is to be

d e te rm in e d a c c o rd in g ly . T h e fo llo w in g fo rm u la is fo r th e c a lc u la tio n o f n itro g e n
c o n c e n tra tio n in fis h p ro te in e x tra c t (see B-1 fo rm u la (1) ):
N c o n te n t (m g N /m l) = (b - a) x 0.1 x 14.00 x 1 /n
b: s a m p le titra tio n v a lu e (ml)
a: b la n k titra tio n v a lu e (ml)
n: m l o f e x tra c t used in d ig e s tio n
B a sed on th e c o n c e n tra tio n o f n itro g e n p ro te in in th e fish p ro te in e x tra c t, a p p ro p ria te

d ilu tio n ca n be m a d e u s in g K C I-p h o s p h a te b u ffe re d s o lu tio n fo r th e p re p a ra tio n o f
c a lib ra tio n curve.
T h e c o n c e n tra tio n o f th e d ilu te d fis h p ro te in e x tra c t s h o u ld fall in b e t w e e n 0.1 to 0.5

m g N /m l.
2. P re p a ra tio n o f p ro te in e x tra c t fo r s p e c tro p h o to m e tric reading.
P re p a re 2 s e ts o f 6 te s t tu b e s , e a ch c o n ta in in g 5 , 4 , 3, 2 , 1 and 0 m l (blank) o f B o vin se ru m

a lb u m in w o rk in g s o lu tio n o r fis h m y o fib rilla r p ro te in e x tra c t and 0 , 1, 2, 3, 4 a nd 5 m l o f
K C l-p h o s p h a te b u ffe re d s o lu tio n re s p e ctive ly. P ip e tte 5 ml each R e agen t A to o n e s e t o f
te s t tu b e s a n d p ip e tte 5 m l e a ch R e a gen t B to th e o th e r se t o f te s t tu b e s . S h ake w e ll and
leave to s ta n d 2 hrs a t ro o m te m p e ra tu re (26°C). S e t u p th e s p e c tro p h o to m e te r as
s p e c ifie d b y th e m a n u fa c tu re r, a d ju s t th e w a v e le n g th to 5 45 nm an d read th e a b s o rb a n c e
o f th e s o lu tio n re la tive to th e re a g e n t b la n k (co n ta in s o n ly K C l-p h o s p h a te b u ffe re d
so lu tio n ).
3. C a lc u la tio n & c a lib ra tio n c u rv e
C a lc u la te th e s o lu tio n s ’ a b s o rb a n c e c o n ta in in g va rio u s c o n c e n tra tio n o f p ro te in s o lu tio n s .
A b s o rb a n c e 545nm = (O .D .A - B la n k A) - (O .D .B - B la n k B)
O .D . a a n d O .D .B = o p tic a l d e n s ity o f s a m p le s o lu tio n s w ith R eagen t A a nd B,
re s p e c tiv e ly .
B la n k A a n d B la n k B = o p tic a l d e n s ity o f b la n k s o lu tio n s w ith R eagen t A a nd B,

re s p e c tiv e ly .
B-2.2
P lo t th e a b s o rb a n c e va lu e s o f th e p ro te in s o lu tio n s ve rsu s th e c o n c e n tra tio n s o f th e
p ro te in s o lu tio n s to o b ta in th e c a lib ra tio n curve.
B. D E T E R M IN A TIO N OF PR O TEIN C O N C E N TR A TIO N OF UNKNO W N SAM PLE (FISH

M Y O F IB R IL L A R P R O TE IN EXTRACT)
P ip e tte 5 m l p ro te in s a m p le a nd 5 m l R e agen t A in to a te s t tu b e . T o a n o th e r te s t tu b e a d d 5
m l p ro te in s a m p le a n d 5 m l R e a g e n t B. A ls o p re p a re a n o th e r 2 te s t tu b e s e ach c o n ta in in g
5 m l K C l-p h o s p h a te b u ffe re d s o lu tio n a n d 5 m l R eagen t A a nd B, re sp e c tiv e ly . S h a ke w e ll
a n d leave to s ta n d a t ro o m te m p e ra tu re fo r 2 hrs. S e t up th e s p e c tro p h o to m e te r, a d ju s t
th e w a v e le n g th to 5 4 5 nm , a nd read th e a b s o rb a n c e o f th e s o lu tio n re la tive to th e re a g e n t
blank.
IV C A L C U L A T IO N S
B a sed on th e c a lib ra tio n c u rve , e x p re s s th e re su lt in m g N /m l. C o n v e rt th e value to

e q u iv a le n t m e a t w t. a n d e x p re s s as m g N /1 0 0 g sa m p le , if req u ire d .
N .B. E x p e rim e n ts have s h o w n th a t th e re su lts are re la tive ly reliable as c o m p a re d to re su lts

o b ta in e d b y K je ld a h ls ’ m e th o d fo r fis h m y o fib rilla r p ro te in e x tra c t in th e c o n c e n tra tio n
ra n g e o f 0 .1 -0 .5 m g N /m l.
REFERENCE
U m e m o to , S. (1966)
A m o d ifie d m e th o d fo r e s tim a tio n o f fish m u s c le p ro te in b y B iu re t m e th o d . B ull. Ja p . S o c.
S ci. Fish. 3 2 :4 2 7 -4 3 5 .
B-2.3
DETERMINATION OF TRIMETHYLAMINE OXIDE (TMAO-N),
TRIMETHYLAMINE (TMA-N), TOTAL VOLATILE BASIC
NITROGEN (TVB-N) BY CONWAY’S METHOD
NG C.S.
INTRODUCTION
Trimethylamine oxide (TMAO) is a nitrogenous compound commonly present in marine
organisms. It has been suggested that TMAO functions as an osmoregulator in these animals.
The degradation of TMAO into simpler compounds such as trimethylamine (TMA), dimethyla
mine (DMA) and formaldehyde (FA) depends on the enzymes present in the tissue.
Generally, TMAO breaks down to TMA in marine fishes, either by endogenous enzymes,
bacteria enzymes or both. However in gadoid fishes, the TMAO is broken down to DMA and FA.
The use of TMA as an index of fish freshness was first proposed by Beatty and Gibbons
(1936). This was based on the observation that the production of TMA was dependent on
bacteria activity, and the role of autolysis was negligible. The source of TMA in ordinary muscle
is due to the bacterial degradation of TMAO to TMA, while in dark muscles, TMA was derived
both from bacteria activity as well as from endogenous enzymes.
In recent years, there are opinions that TMA itself may not be a very suitable freshness
index. This is because the TMA content in a fish may vary with season, and also, the distribution
of TMA within a piece of fillet may not be uniform. Under the local conditions, TMA was found to
be a good indicator of freshness for white pomfret, Chinese pomfret, grouper and siakap. TMA
is not a good indicator of freshness for lizard fish. Instead, DMA and FA are suitable indices.
The total volatile basic substances (TVB) in fish meat is mainly composed of ammonia,
TMA, and DMA. The level of TVB increases after spoilage begins (both enzymatic and bacterial).
It does not distinguish the origin nor component of these volatile compounds, hence its use is
more general.
In this laboratory, the microdiffusion method devised by Conway is adopted. In this

method, TMA, TMAO and TVB are determined as their nitrogen. To obtain the actual amount of
TMA or TMAO the nitrogen values must be divided by the amount of nitrogen present per
molecule of TMA or TMAO.
PRINCIPLE OF THE CONWAY UNIT IN DETERMINATION OF TVB

The solution in the inner ring of the Conway unit contains a 1% solution of boric acid with
bromocresol green and methyl red indicator. The sample extract is in the outer ring. On the
addition of K2CO3 the sample extract becomes alkaline. The TMA and related compounds
present in the sample extract are released in alkaline condition as volatile compounds. The
volatile compounds diffuse into the boric acid solution to form boric acid salt of these
compounds. These salts are reduced to HCl-salts by a strong acid (HCl) during titration.
B-3.1
I REAGENTS
a) Inner ring solution — 1% boric acid solution containing indicator:
Take 10 g of boric acid in 1 litre flask, add 200 ml of ethanol. After dissolving boric acid,
add 10 ml of mixed indicator solution, then make up to 1 litre with distilled water.
b) Mixed indicator solution:

Dissolve bromocresol green (BCG) 0.01 g and methyl red (MR) 0.02 g in 10 ml of ethanol.
c) 0.02N HCl:
Dilute 20 ml of 1N HCl standard solution with distilled water and make up to 1000 ml.
d) Saturated K2CO3 solution:

Take 60 g of potassium carbonate (K2CO3), and add 50 ml of distilled water. Boil gently for
10 min. After cooling down, obtain filtrate through filter paper.
e) 50% K2CO3 solution:

Dilute saturated K2CO3 solution twice with distilled water.
f) 4% trichloroacetic acid (CCl3COOH) (TCA) solution:

Dissolve 40 g of TCA in 960 ml of distilled water.
g) Sealing agent:
Take 3 g of Tragacanth gum, add 30 ml of distilled water, 15 ml of glycerine and 15 ml of
50% saturated K2CO3 solution and mix well.
h) Neutralized 10% formaldehyde solution:

Add 10 g of MgCO3 to 100 ml of formalin (35% formaldehyde solution) and shake in order
to neutralize the acidity of formalin. Filter and dilute filtrate 3 times with distilled water.
i) 1% TiCl3 aqueous solution:

Take 6.7 ml of 15% TiCl3 solution into 100 ml volumetric flask and make up to 100 ml with
distilled water.
j) Saturated KNO3 aqueous solution:

Dissolve about 55 g of KNO3 in 50 ml of distilled water.
II APPARATUS
Conway’s unit:
Wash with detergent (use neutral detergent if available), then rinse with running water and
leave until dry. Do not wipe with cloth.
Micro-burette
B-3.2
Ill PROCEDURE
A. SAMPLE EXTRACTION
1. Take 2 g of fish meat in a mortar and grind well.
2. Add 8 ml of 4% TCA solution and grind well.
3. Stand for 30 min at ambient temp. with occassional grinding.
4. Filter through filter paper (Whatman No. 41). (or Centrifuge at 3000 rpm, for 10 min.)
5. Keep the filtrate in -20°C freezer if necessary.
B. DETERMINATION OF TVB-N
1. Apply sealing agent to Conway’s unit.
2. Pipette 1 ml of inner ring solution into inner ring.
3. Pipette 1 ml of sample extract into outer ring.
4. Slant the Conway’s unit with cover.
5. Pipette 1 ml of saturated K2CO3 solution into outer ring.
6. Close the unit.
7. Mix gently.
8. Stand for 60 min. at 37°C in incubator.
9. Titrate inner ring solution with o.o2N HCl using a micro-burette until green colour turns
to pink.
10. Do blank test using 1 ml of 4% TCA instead of sample extract.
C. DETERMINATION OF TMA-N
Principle of TMA-N determination is similar to TVB-N determination except addition of
formaldehyde to the sample solution. Formaldehyde is added in order to fix any ammonia
present in the sample.
1. Apply sealing agent to Conway’s unit.
2. Pipette 1 ml of inner ring solution into inner ring.
3. Pipette 1 ml of sample extract into outer ring.
4. Pipette 1 ml of neutralized 10% formaldehyde into outer ring.
5. Slant the Conway’s unit with cover.
6. Pipette 1 ml of saturated K2CO3 solution into outer ring.
7. Close the unit.
8. Mix gently.
B-3.3
9. Stand fo r 60 min. at 37°C in incubator.
10. Titrate inner ring solution with 0.02N-HCI using a micro-burette until green colour turns
to pink.
11. Do blank test using 1 ml of 4% TCA instead of sample extract.
CALCULATION OF TMA-N OR TVBN
TMA-N or Amt. of HCl Amt. of ammonium Ratio of the amount

TVBN used in x nitrogen equivalent to x of sample used to
(mg/100 g) titration 1 ml of 0.02N HCI 100 g muscle
M
( WS x + VE x 100
100
— (VS VB) x (NHCl
– x A N) x
WS
where
VS = Titration volume of 0.02N HCI for sample extract (ml)
VB = Titration volume for blank (ml)
N HCl = Normality of HCI (=0.02N x f, factor of HCI)
An = Atomic weight of Nitrogen (x 14.00)
WS = Weight of muscle sample (g)
M = percentage moisture of muscle sample.
VE = Volume of 4% TCA used in extraction
NOTE: 1 ml of 0.02N HCI = 0.28 ammonium nitrogen
= (NHCl x f x 14.00)
D. DETERMINATION OF TMAO-N
Reduction of sample extract

1. Take 2 ml of the filtrate (sample extract) or its dilute into a test tube.
2. Add 1 ml of 1% TiCI3 and fully mix, then confirm that pink colour does not disappear.
3. Stand in an 80°C water bath for 90 sec.
4. Add saturated KNO3 dropwise until the pink colour disappears.
5. Cool in water.
B-3.4
6. Transfer the solution to 10 ml volumetric flask.
7. Make up to 10 ml with distilled water.
8. Proceed as for TMA-N determination.
CALCULATION OF TMAO-N
TMAO-N = (TMA-N after TiCl3 reduction)* — (TMA-N before TiCl3 reduction)
* Care must be taken to obtain the correct dilution factor since in TMAO-N determination, the
sample was made up to 10 ml before applying into the Conway unit.
ANNEX I. SCHEMATIC FORM FOR PREPARATION OF REAGENTS
REAGENTS
a) Inner ring solution
b) 0.02N-HCl (Accurate)
Dilute 20 ml of 1N-HCl with water and make the volume to 1 litre.
c) Saturated potassium carbonate
d) 50% saturated potassium carbonate

Dilute c) solution with water (1:1).
B-3.5
e) 10% TCA
Dissolve 100 g of trichloroacetic acid (CCl3COOH) in 900 ml of water.
f) 5% TCA
Dilute e) solution with water (1:1).
g) Sealing agent
ANNEX II. SCHEMATIC FORM FOR PREPARATION OF SAMPLE FOR TVB-N AND TMA
B-3.6
ANNEX III. PROCEDURE FOR TVB-N DETERMINATION
Do blank test using 1 ml 4% TCA instead of sample.
B-3.7
ANNEX IV. PROCEDURE FOR TMA-N DETERMINATION
Do blank test using 1 ml 4% TCA instead of sample.
REFERENCES
Beatty, S.A. and N.E. Gibbons (1936)
The measurement of spoilage of fish. J.Biol.Bd. Can. 3:77-91.
Conway, E.J. and A. Byrne (1936)

An absorption apparatus for the micro-determination of certain volatile substances. I. The
micro-determination of ammonia. Biochem. J. 27:419-429.
B-3.8
DETERMINATION OF DMA-N BY DYER’S COLORIMETRIC
METHOD USING COPPER DITHIOCARBAMATE
NG C.S.
INTRODUCTION
The precursor of dimethylamine (DMA) in fish meat is trimethylamine oxide (TMAO). In the
gadoid species, TMAO present in ordinary muscle is decomposed to formaldehyde (FA) and
DMA simultaneously. This is usually attributed to endogenous enzymes. In the tropics, lizard
fish is known to show a similar breakdown sequence. In fresh fish, and fish in the early stages of
spoilage, the amounts of primary amines is low. The main secondary amine present is DMA.
Hence measurement of DMA can be used as a spoilage indicator. However, at the later stages
of spoilage, numerous other secondary amines are formed, and these will interfere with the
results of the Dyer’s colorimetric method.
In the laboratory determination of TMA-N and TMAO-N, the presence of DMA interferes
and yields a higher value for the parameters measured. If the true amounts of TMA-N and
TMAO-N are desired, the interference due to DMA must be discounted.
DMA and other secondary amines can react with nitrite salts to form dimethylnitro
soamine, a known carcinogen. Therefore, it is important to determine the amount of DMA
present in fish and other food.
PRINCIPLE OF DYER’S COLORIMETRIC METHOD

Volatile secondary amines such as dimethylamine, di-n-propylamine etc. reacts with
carbon disulfide to form dialkyl-dithiocarbamic acid (Equation 1). This dialkyl-dithiocarbamic
acid reacts with NH4+ or Na+ to form dialkyl-dithiocarbamate (Equation 2). Dialkyl
dithiocarbamate chelates with Cu2+ to form a yellow complex, Cu-dialkyl-dithiocarbamate
(Equation 3).
(1
)
(2)
(3)
B-4.1
I REAGENTS
All reagents should be of GR grade.
a) 5% (v/v) carbon disulfide-toluene solution

Mix 5 ml of carbon disulfide with 95 ml of toluene.
b) Copper-ammonium reagent
Dissolve 25 g of ammonium acetate and 0.2 g of cupric sulfate in 30 ml of distilled water,
and mix this solution with 25 ml of 40% NaOH. To this add 20 ml of cone, ammonia (s.g.
0.88-0.90) and mix well, then make up to 100 ml with distilled water.
c) 30% acetic acid.
d) Anhydrous sodium sulfate.
e) DMA standard stock solution.

Take 60.0 mg of DMA-HCl salt into 100 ml volumetric flask and make up with distilled
water. This solution contains about 0.1 mg DMA-N/ml.
f) DMA standard working solution

Take 10 ml of DMA stock solution into 100 ml of volumetric flask and make up with 2%
TCA solution. This solution contains about 10 ug DMA-N/ml.
II PROCEDURE
A. SAMPLE PREPARATION
1. Take 5 g of sample in a mortar and grind well.
2. Wash the sample into a 100 ml volumetric flask with about 50 ml of distilled water.
3. Stand for 10 min after stirring well.
4. Add 8 ml of 25% TCA and mix well.
5. Make up to 100 ml with distilled water and mix well.
6. Stand for 30 min at ambient temperature.
7. Filter the solution with filter paper (Whatman No. 41).
B. DETERMINATION OF DMA
The following procedure should be done in a fume cupboard.
1. Take 5 ml of the filtrate in a test tube with stopper.
2. Add 1 ml of copper-ammonium reagent and mix.
3. Add 10 ml of 5% CS2-toluene solution and then stopper the test tube.
4. Stand for 2 min in 50°C water bath.
B-4.2
5. Shake for 1 min.
6. Add 1 ml of 30% acetic acid.
7. Shake for 20-30 sec.
8. Stand for 10 min at ambient temperature.
9. Transfer the toluene layer to another tube containing about 0.5 g of anhydrous Na2SO4
after the toluene layer becomes clear.
10. Measure the absorbance at 440 nm.
11. Repeat the procedure with 5 ml of 2% TCA as blank.
C. STANDARDISATION OF DMA STANDARD SOLUTION

Principle:
(1)
(2)
(3)
1. Take 10 ml DMA stock solution (DMA.HCl) into distillation tube. Add 20 ml distilled water
and 6 ml 10% NaOH.
2. Steam distill to vaporise the DMA, and absorb into 20 ml of 4% H3BO3.
3. Titrate DMA.H3BO3 solution with 0.05N H2SO4 using methyl red-bromocresol green
mixed indicator.
4. Calculate the factor of DMA. HCl standard solution as:-
B-4.3
D. PREPARATION OF CALIBRATION CURVE
1. Take 0.4, 0.8, 1.2, 1.6 and 2.0 ml of DMA standard working solution into the test tube
(volume about 40 ml), and add 4.6, 4.2, 3.8, 3.4 and 3.0 ml of 2% TCA solution,
respectively. These solutions contain 4, 8, 12, 16 and 20 μg DMA-N, respectively.
2. Repeat the procedure for determination of DMA.
E. CALCULATION OF DMA-N CONTENT OF SAMPLES

1. Obtain the amount of DMA-N (A μg) contained in 5 ml of sample solution from the
calibration curve.
2. DMA-N (mg/100 g)
where W = weight of meat

f = factor of DMA standard solution
d = dilution factor (if any)
REFERENCE
Dyer W.J. and Y.A. Mounsey (1945). Amines in fish muscle. II. Development of trimethylamine
and other amines. J. Fish. Res. Bd. Can. 6(5):359-367.
B-4.4
DETERMINATION OF FORMALDEHYDE IN FISH MEAT
USING NASH’S REAGENT
NG C.S.
INTRODUCTION
It has been postulated that the enzymatic degradation of trimethylamine oxide (TMAO)
results in the simultaneous formation of dimethylamine (DMA) and formaldehyde (FA). This
phenomena had been reported to have a correlation of 0.89. (Amano et al, 1963). FA and
DMA formation occurs widely in the gadoid species. In the tropical areas, lizard fish (Saurida
sp) also exhibits this trend.
Formaldehyde reacts quickly with muscle tissues, causing protein denaturation. Forma
tion of FA is accelerated by freezing.
In this method of FA determination, FA is reacted with an ammonium salt and

acetylacetone under neutral conditions to form diacetyldihydrolutidine (DDL). DDL is a yellow
compound with maximum absorbance at 412 nm.
MOLECULAR FORMULA AND REACTION
I REAGENTS
a) Acetylacetone reagent (Nash’s reagent)
Dissolve 150 g of ammonium acetate, 3 ml of acetic acid and 2 ml of acetylacetone in
distilled water and make up to 1 litre.
b) Formaldehyde standard stock solution

Pipette 0.3 ml of 35% formaldehyde and fill up to 100 ml to get approximately 1,000 ppm
solution in distilled water. This aqueous solution is stable for several months.
c) Formaldehyde standard stock solution.

Dilute the stock solution 100 times as follows:-
Pipette 10 ml of the stock solution (approximately 1,000 ppm) and make up to 100 ml with
B-5.1
distilled water to get approx. 100 ppm solution. Ten ml of 100 ppm solution is diluted 10
times with distilled water in the volumetric flask. This final dilute gives approx. 10 ppm
solution of formaldehyde. This dilute is not stable, so, it is necessary to be renewed in each
series of determination.
d) 0.1N Sodium thiosulfate standard solution

Dissolve 25 g of Na2S2O3.5H2O in distilled water which is cooled after boiling, and make
up to 1 litre. Standardize after standing 1 to 2 days by the procedure described in the
determination of peroxide value (C-5).
e) Sodium bisulfite solution (approximately 0.1N)

Dissolve 5.2 g of NaHSO3 in distilled water and make up to 1 litre.
f) Iodine solution (approximately 0.1N)

Dissolve 12.7 g of l2 and 40 g of Kl in 25 ml of distilled water and make up to 1 litre.
g) 1.5% Starch solution

Weigh 1.5 g of starch and add 100 ml of distilled water, then boil the solution for 30 sec.
STANDARDISATION OF FORMALDEHYDE SOLUTION

1. Pipette 5 ml of formaldehyde solution, approximately 1,000 ppm, into a 200-300 ml conical
flask and add 50 ml of distilled water.
2. Add 10 ml of 0.1N sodium bisulfite solution, and let it stand for about 30 min with
occasional shaking.
3. Add iodine solution until the colour turns brown. Note the volume of iodine used. Titrate
the excess iodine with sodium thiosulfate standard solution until just before the yellow
colour disappears. Add 1 ml starch solution as indicator, and continue titration until the
dark-blue colour disappears. The volumes of iodine solution and thiosulfate solution are
noted.
4. Calculate the factor of formaldehyde standard solution.
The following reactions occur.
(1
)
(2)
(3)
moles Na2S2O3 = 1 mole NaHSO3 = 1 mole HCHO
B-5.2
The specific gravity of 35% formaldehyde at 25°C is 1.08. The molarity of the 1000 ppm
solution is 0.032375. Since 1 mole of formaldehyde is equivalent to 2 moles of sodium
thiosulfate, using N1 V1 = N2 V2,
N1, Normality of formaldehyde = factor x Molarity x equivalent

= f x Mx e
= f x 0.03237 x 2
= Volume of formaldehyde = 5 ml
N2 = Normality of Na2S2O3 = 0.1
V2 = Volume of Na2S2O3 = (Vol titrated in sample) - (Vol titrated in blank).
= Vs - VB
II APPARATUS AND INSTRUMENTS

Hitachi Spectrophotometer (λ = 412 nm)
Beckman Model 3560 digital pH meter
Yamato ultra disperser
beakers (50 ml)
long test-tubes (15ml)
burette (25 ml)
Pipetman micropipette (max vol= 1 ml)
filter paper (Whatman No. 41, Ø 15 cm)
III PROCEDURE
A. SAMPLE PREPARATION
1. Weigh 5 g of minced meat accurately in a 30-50 ml beaker.
2. Add 20 ml of 5% TCA solution and homogenize well with homogenizer.
3. Stand in an ambient temperature for 30 min.
4. Filter the supernatent with filter paper, Whatman No. 41.
5. Add 10 ml of 5% TCA solution to the residue, homogenize again, then filter.
6. Neutralize the combined filtrate to pH 6.0-6.5 by using pH meter with 1N or 0.1N KOH
dropwise, and make up to 50 ml with distilled water.
B-5.3
B. DETERMINATION OF FORMALDEHYDE
1. Take 3 ml of the neutralized filtrate in a test tube, add 3 ml of the acetylacetone reagent
and mix well.
2. Stand in water bath (60°C) for 15 min.
3. Cool the solution in running water.
4. Measure the absorbance of the solution against the blank solution at 412 nm (Blank
solution contains distilled water instead of the neutralized filtrate).
C. PREPARATION OF CALIBRATION CURVE

1. Pipette 0, 0.3, 0.6, 1.2 and 2.4 ml of 10 ppm formaldehyde standard working solution into
test tube using micro-pipette. These solutions contains 0, 3, 6, and 12 and 24 μg of
formaldehyde, respectively.
2. Add 3, 2.7, 2.4, 1.8 and 0.6 ml of distilled water, respectively, then add 3 ml of the
acetylacetone reagent.
3. Continue as in the procedure B) 2. to 4. after mixing well.
IV CALCULATION OF FORMALDEHYDE CONTENT
Formaldehyde = A (Total make up vol. of filtrate) x f

X
(µg/g) (Vol. of filtrate used) (Weight of sample)
where A = Reading from calibration curve (μ g)

f = factor of formaldehyde of standard solution.
REFERENCES
Amano K., K. Yamada and M. Bito 1963A.
Detection and identification of formaldehyde in gadoid fish. Bull. Jap. Soc. Sci. Fish.
29(7): 695-701.
Amano K., K. Yamada and M. Bito 1963B,

Contents of formaldehyde and volatile amines in different tissues of gadoid fish. Bull. Jap.
Soc. Sci. Fish. 29(9): 860-864.
Hebard C.E., J.F. George and R.E. Martin.

Occurence and significance of trimethylamine oxide and its derivatives in fish and shellfish.
In: Martin R.E. et al (Editors) 1982. Chemistry and biochemistry of marine food products
p: 164-165. Publ. AVI., Westport.
B-5.4
DETERMINATION OF K VALUE
NG C.S.
INTRODUCTION
The K value is an index to measure the enzymatic freshness of fish and squids.
Immediately after death, ATP (adenosine triphosphate) and related com pounds are broken
down by endogenous enzymes. A typical schematic breakdown can be represented as:-
ADP = adenosine diphosphate

AMP = adenosine monophosphate
IMP = inosine monophosphate
HxR = inosine or hypoxanthine riboside
Hx = hypoxanthine
The K value is defined as
[HxR] +[Hx]
K% = x 100
[ATP] + [ADP] + [AMP] + [IMP] + [HxR] + [Hx]
Ideally, the K value should be measured before exogenous enzymatic activities such as
bacterial enzymes begin. In applying K value, care should be exercised to ensure that it is
reliable. For example, the K value of a processed fillet may be higher as water soluble
components such as IMP may have been washed away. Sampling the unexposed meat will
prevent such an error. Skins and dark muscles of fish should be excluded during sampling.
Guanine found in the skin will be eluted with the hypoxanthine fraction while dark muscles have
a high inosine content.
The present method cannot be directly used for measuring the K value of squids. In the
squid, the AMP breaks down directly to HxR. Separation of AMP and HxR is more difficult
compared to separation of IMP and HxR. A modified method as proposed by Uchiyama (1984)
should be adopted.
PRINCIPLE OF ION EXCHANGE CHROMATOGRAPHY

Ion exchange chrom atography distinguishes one component in a mixture from another on
the basis of the number of charges of appropriate sign available on each molecule for
interaction with the ion exchanger under the conditions imposed. Molecular size is an important
factor and the distribution of charges also plays a role.
Ion exchangers can be classified into tw o categories:-
(i) those that bear positive charges and are called anion exchangers because they interact
with anions.
(ii) those that bear negative charges and are called cation exchangers because they interact
with cations.
In the present method, an anion exchanger (Cl- form) is used. Uchiyama et. al (1972) had
reported that using authentic mixtures of ATP and its related compounds charged with Dowex
B-6.1
1-X4 column, the elution of HxR and Hx takes place in the region of pH 6.0 and 0.1M NaCl,
while nucleotides such as ATP, ADP, AMP and IMP are eluted at a acidity of less than pH3, and
within the range of up to 0.15M NaCl.
I PREPARATION OF SAMPLE
One gram of ordinary muscle from fish is sufficient. Care should be taken to exclude red
muscle, fibrous tissues and skin. Sample treatment procedure is illustrated in Scheme 2.
II REAGENTS
A. FOR SAMPLE PREPARATION
All reagents listed here should be kept at 5°C until used.
a) 10% perchloric acid (PCA): Dissolve 10 g of PCA (60-70%, HClO4) in 90 ml of distilled

water.
b) 5% PCA: Dissolve 10 g of PCA in 190 ml of distilled water.
c) Neutralized PCA: Neutralize 100 ml of 5% PCA to pH 6.4 with 10N-KOH using pH meter,
then filter precipitates (KClO4) through filter paper after cooling the neutralized PCA at 5°C.
d) 10N-KOH: Dissolve 56 g of potassium hydroxide (KOH) in distilled water and make up to

100 ml.
e) 1N-KOH: Dissolve 5.6 g of KOH in distilled water and make up to 100 ml.
B. FOR ION-EXCHANGE CHROMATOGRAPHY

a) 0.5M NH4OH solution: Dilute 4 ml of 25% NH4OH with 96 ml of distilled water.
b) Solution A = 0.001 N HCl: Dilute 1 ml of 1N HCl standard solution to 1000 ml with distilled
water.
c) Solution B = 0.01N HCl containing 0.6M NaCl: Dissolve 35.07 g of NaCl in distilled
water, then mix this NaCI solution with 10 ml of 1N HCl standard solution and make up to
1000 ml with distilled water finally.
d) Anion exchange resin: AG (R) 1-X4, 400 mesh Cl (chloride)-form (Bio-Rad Co.).
C. PREPARATION OF ION-EXCHANGE RESIN (SCHEME 1)

a) Acetone
b) 0.1N NaOH
c) 0.1N HCl
III APPARATUS
Chromatography System
Figs. 1 and 2 show two systems for simplified method estimation of K-value.
Column
As shown in Fig 3, use the column (inner Ø 6 mm) with coarse glass filter at the bottom
part. The height of resin is around 50 mm.
B-6.2
IV PROCEDURE
A. PREPARATION OF ION-EXCHANGE RESIN
See Scheme 1.
B. PREPARATION OF SAMPLE EXTRACT

See Scheme 2.
C. CHROMATOGRAPHY (Also see Scheme 3)

1. Take 2 ml of neutralized muscle extract in a test tube.
2. Adjust pH to 9.4 by using pH test paper, with a few drops of 0.5M NH4OH.
3. Apply it onto the column.
4. Wash the inside wall of the column with a few ml of distilled water.
5. In system 1 (Fig 1), onto a column attach a siphon tube which is set in a beaker containing
20 ml of distilled water. In system 2 (Fig 2), attach a separating funnel instead of a siphon
onto the column and pour 20 ml of distilled water into the separating funnel.
6. Wash out unabsorbed ultraviolet-absorbing-compounds with distilled water from the

column.
7. Pour 45 ml of solution A into the beaker or the separating funnel to elute hypoxanthine
riboside (HxR) and hypoxanthine (Hx).
8. Collect the eluate in a 50 ml volumetric flask. Maintain the flow rate at 1-1.5 m l/m in.
9. After all the solution A had passed into the resin, run 45 ml of solution B into the column to
elute ATP, ADP, AMP and IMP.
10. Collect the eluate in another 50 ml volumetric flask.
11. Make up the eluates to 50 ml with solutions A and B, respectively.
12. Measure the absorbance of the tw o eluates at 250 nm.
V CALCULATION
E250nm A
K(%) = x 100
E250n A + E250nm B
where E250nm A: [OD at 250nm of the solution A-eluate]

-[O D at 250nm of the soln A];
E250nm B: [OD at 250nm of the solution B-eluate]
- [OD at 250nm of the soln B] .
B -6.3
Fig 1. One system for simplified estimation
of the K-value using a
peristaltic pump (system 1).
Fig 2. Another system for the estimation Fig 3. Column

of K-value using a
separating funnel (system 2).
B-6.4
SCHEME 3. SIMPLIFIED FRACTIONATION METHOD FOR K-VALUE
REFERENCES
Ehira S., H. Uchiyama, F. Uda and H. Matsumiya. (1970).
A rapid method for determination of acid soluble nucleotides in fish muscle by concave
gradient elution. Bull. Jap. Soc. Sci. Fish. 36:391-496.
Uchiyama H., Ehira S., and Kato N. (1972).

Analytical methods for estimating freshness of fish. In Okada et al (Eds). Utilization of marine
products. Publ: Overseas Technical Cooperation Agency Govt. of Japan.
Uchiyama H. (1978).
Analytical method for estimating freshness of fish. Training Dept. SEAFDEC: 1-9.
Uchiyama H. and Kakuda K. (1984).

A simple and rapid method for measuring K value, a fish freshness index. Bull. Jap. Soc. Sci.
Fish. 50(2):263-267.
B-6.7
SCHEME 1. PREPARATION OF ION-EXCHANGE RESIN
* Repeat washing with distilled water until filtrate (water) is neutral. Activated resin is stored
at 5°C under water.
B-6.5
SCHEME 2. PREPARATION OF FISH MUSCLE EXTRACT
1 g fish meat and Centrifuge at 2,000/3,000 rpm,

2 ml 10% PCA (chilled) 0-4°C, for 2-3 min
↓
— grind Decant supernatant into
↓ 10 ml volumetric flask
Centrifuge at 2,000/ ↓
3,000 rpm for 2-3 min Precipitate (KCIO4) and
2 ml neutralised PCA
↓
Decant supernatant (pH 6.4, chi led)
(filter to remove fat if necessary)
↓ —grind
Residue and 2 ml 5% ↓
PCA (chilled) Centrifuge at 2,000/
3,000 rpm for 2-3 min
— grind ↓
Decant supernatant into the
Centrifuge at 2,000/ 10 ml volumetric flask
3,000 rpm for 2-3 min ↓
Supernatant collected in the
↓
Decant supernatant 10 ml volumetric flask
↓ ↓
Residue and 2 ml 5% Make up to 10 ml mark with
PCA (chilled) neutralised PCA (pH 6.4, chilled)
Jr
— grind Transfer into 14 ml Bijou bottle
and store frozen at -2 0 °C if
Centrifuge at 2,000/ necessary.
3,000 rpm for 2-3 min ↓
For application to ion exchange
Decant supernatant column
↓
Extracted sam ple, ~ 6 ml
(Pooled supernatant)
↓
Neutralise with
10N KOH (~ 6-8 drops)
Adjust to pH 3,
test with TB paper
(TB = Thymol Blue)
↓
Neutralise with
1N KOH (~ 4 drops)
Adjust to pH 6.5-6.8
Test with BTB paper
BTB = Bromothymol Blue)
B—6.6
FRESHNESS TESTING PAPER
NG C. S.
INTRODUCTION
Measuring K value by means of ion exchange chromatography and spectrophotom etry is

tedious and cumbersome. The Freshness Testing Paper (FTP)* technique, aims at practicality
and suitability for use in the field. The principles involved utilise enzyme actions to convert
inosine (HxR) and hypoxanthine (Hx) to uric acid, which changes the colour of the dye present
in the paper.
This method should preferably be used after it had been calibrated against the ion
exchange chrom atographic method. The enzymes present in fishes may vary from species to
species, and calibration should be conducted for each species. For very crude estimation, no
calibration is required.
Since the FTP uses enzymes, storage of the test paper at low temperature is essential
(preferably at -6 0 °C ) to ensure the functionality of the enzymes. This is one of the major
disadvantage of this technique.
PRINCIPLE OF FRESHNESS TESTING PAPER

This technique uses enzymatic degradation and the subsequent colour conversion of a
redox dye to indicate “ freshness”
HxR + Hx
The K value (%) is defined as x 100
ATP + ADP + IMP + AMP + HxR + Hx
In the FTP, the enzymes nucleoside phosphorylase and xanthine oxidase are embedded in
the test paper. On application of the sample extract, the following reactions occur.
↔
HxR + Pi Hx + ribose-l-phosphate
nucleoside
phosphorylase
Hx + O2 → xanthine + H2O 2
xanthine
oxidase
xanthine + O 2 → uric acid + H2O 2

xanthine
oxidase
The uric acid formed changes the colour of the redox dye present in the FTP. The colour
intensity is proportional to the content of HxR + Hx.
* The FTP Test Kit is patented and sold by Kankyo Bunseki Centre K.K. Tokyo.
B- 7.1
I PROCEDURE
1. Take 0.5 g minced fish meat and add 0.5 g treated sand. Grind well in a small mortar.
2. Add 1.5 ml of FB* solution. Grind well. Add another 3.5 ml FB solution and grind well.
3. Dip FTP into homogenised solution. Remove and blot off excess solutions on filter paper.
Keep the FTP in a plastic bag (transparent; and keep at room temperature for 10-15 min.
(Standardise the time for each species).
4. Compare the colour (red to purple) of FTP with the colour chart provided. Read the
corresponding K value.
STANDARDISE FTP TO K VALUE BY ION-EXCHANGE CHROMATOGRAPHY

1. Samples of the species under study of varying freshness is required.
2. Meat samples were individually prepared. A portion of the meat is used in the FTP, while
the corresponding portions are subjected to conventional K value analysis.
3. The colour intensity and the corresponding K value are correlated. Care should be taken to
standardise the time of reading of the coloured strips of FTP.
PRECAUTIONS
1. All the materials supplied with the FTP kit are easily degraded, and should be stored frozen
(-60°C preferable) and in the dark. Anaerobic conditions will prolong the shelf life.
2. Check the expiry date before use. Expired products will give unreliable results.
3. The resulting colour is unstable in light, and will eventually fade. Readings should be
conducted immediately after the full colour had developed.
The procedure for FB solution preparation as stated in the test kit.
B -7.2
ANALYSIS OF OILS
SIGNIFICANCE OF ANALYSIS OF LIPIDS
LOW L .K . & NG C .S .
Fish lip id s e x is t as p h o s p h o lip id s (tissue fat) and trig ly c e rid e s (d e p o t fa ts o r neu tra l lipids).
D u ring s to ra g e , fis h lip id s d e te rio ra te b y h y d ro ly s is a nd o x id a tio n .
B o th p h o s p h o lip id s a n d trig ly c e rid e s are h yd ro lyze d b y en zym e s in to fre e fa tty a cid s. T he

in d ic e s u se d fo r m e a s u rin g th e d e g re e o f h y d ro ly s is are:
i) th e p h o s p h o lip id c o n te n t
ii) th e a c id v a lu e (AV)
iii) th e fre e fa tty a c id v a lu e (FFA)
iv) th e s a p o n ific a tio n v a lu e (SV)
D u ring o x id a tio n , th e h ig h ly u n s a tu ra te d fa tty a c id s o f fish fa ts re a c t w ith a tm o s p h e ric

o xyg e n to y ie ld a c o m p le x se rie s o f c o m p o u n d s in c lu d in g a ld e h yd e s, ke to n e s, a nd a c id s o f
lo w e r m o le c u la r w e ig h t. T h e s e b y -p ro d u c ts c o n trib u te to th e u n p le a sa n t ta s te a nd ra n c id o d o u r
o f s p o ilt fish . E xp o su re o f th e fis h to h e a t a nd light, to m o istu re , a nd to th e p re s e n c e o f tra c e s o f
ce rta in m e ta ls (eg. c o p p e r, n icke l a nd iron) a c c e le ra te s th is o x id a tio n re a ctio n . T h e re a ctio n
in vo lve d m ay be s u m m a riz e d as fo llo w s :-
C h e m ic a l p a ra m e te rs w h ic h are u sed fo r d e te rm in in g th e e x te n t o f s p o ila g e d u e to

o x id a tio n o f fish lip id s a re :-
i) th e p e ro x id e va lu e (POV)
ii) th e th io b a rb itu ric a c id n u m b e r (TBA)
iii) th e o x id a tio n in d e x
C -1 .1
EXTRACTION OF LIPIDS (MODIFIED FOLCH’S METHOD)
LOW L. K. & NG C. S.
INTRODUCTION
A mixture of chloroform and methanol in the ratio of 2:1 (v/v) extract lipid more
exhaustively from animal tissues than most other simple solvents systems. With most tissues,
the lipids are removed almost completely after two or three treatments with the mixture. Most of
the contaminating compounds in the extract can be removed from the chloroform-methanol
(2:1 v/v) mixtures simply by shaking the combined solvents with a quarter of the total volume of
water. The lower phase which comprises 60% of the total volume contains the purified lipid.
This extraction yields approximately a 95-99% recovery of lipids.
The fish sample is chopped into a mince. Depending on the tissue, the following
approximate sample sizes are used.
i) ordinary muscle (20 - 50 g)

ii) dark muscle (15 g)
iii) skin (10g)
II APPARATUS
Homogenizer with ice jacket.
Buchner flask and funnel
Vacuum pump
Nitrogen gas
Separating flasks (1000 ml)
Volumetric flasks (50 ml)
Measuring cylinders (100 and 250 ml)
Whatman No. 1 filter paper (qualitative, 7 cm )
III REAGENTS
a) Purified and distilled chloroform
Wash chloroform once with concentrated sulphuric acid (10 ml H2SO4 for 1 litre of
chloroform). Then wash 2 to 3 times with distilled water using a separating funnel. Collect
washed chloroform and add anhydrous calcium chloride. Stand overnight, then transfer to
distillation flask. Distil and collect fraction which distills over at 60.5°C. Add purified and
distilled methyl alcohol (1 % by volume) as stabilizer. Keep in dark. Should be used within
one month.
b) Purified and distilled methyl alcohol

Add granular potassium hydroxide to methyl alcohol to remove acids, aldehydes and
moisture. Distill and collect fraction which distills over at 64.5°C. Keep in the dark. Should
be used within one month.
c) Chloroform-methyl alcohol (C-M mixture)

Mix reagent from a) with b) in the proportion of 2:1 (v/v).
C-2.1
d) 1% BHA-BHT antioxidant solution
Dissolve 1 g of butylated hydroxyanisole (BHA) and 1 g of butylated hydroxytoluene (BHT)
in 100ml C-M mixture.
e) Anhydrous sodium sulphate
IV PROCEDURE
1. Weigh the chopped sample into the homogenizer cup.
2. Add C-M mixture volume of about 3.5 times the weight of sample, and 2-3 drops of
antioxidant solution.
3. Homogenize for 1 min and filter with Whatman No. 1 filter paper using a Buchner funnel
and vacuum pump.
4. Transfer the residue into the cup and repeat homogenization twice.*
5. Transfer the combined filtrate into a separating flask.
6. Pour distilled water, volume approximately a quarter of that of the extract, into the
separating flask.
7. Shake very gently 2-3 times, and stand overnight.**
8. Drain off the chloroform phase through a Whatman No. 1 filter paper into an Erlenmeyer
flask containing about 2-5 g anhydrous sodium sulphate. Shake well and leave for about 5
min. Decant into an evaporating flask.
9. Wash the filter paper 2-3 times with C-M Mixture.
10. Concentrate the extract with a rotary evaporator under reduced pressure at 40°C
(water-bath temperature).***
11. Dissolve the concentrated extract with C-M Mixture and transfer to 50 ml volumetric flask
using a pipette.
12. Make up to the mark with C-M Mixture.
13. Flush with nitrogen gas and store at -20°C. This sample is used for other tests unless
otherwise specified.
* Not necessary to add antioxidant solution.

** When mixture does not separate well, centrifuge at 8,000 rpm for 10 min.
*** Do not allow to evaporate to dryness.
N.B. Residual water vapour cannot be removed completely.
REFERENCES
Christie, W.W. (1982). In: Lipid analysis (2nd Ed.) Pergamon Press:22
Folch et al. (1951). Journal of Biological Chemistry, 191:833.
C-2.2
DETERMINATION OF TOTAL LIPID CONTENT
INTRODUCTION
This method enables the total lipid of the fresh fish sample to be determined without the
destruction of the lipid extract.
I APPARATUS
Analytical balance (at least 1 mg sensitivity)
Rotary vacuum pump (max vacuum = 3x1 0 -2 mbar)
Water bath with temperature control system (40°C)
Desiccator
Test-tubes
Pipette (5 ml)
II PROCEDURE
1. Dry test-tube in desicator for half an hour and weigh accurately.
2. Pipette accurately 5 ml of the extract into the dry preweighed test-tube.
3. Remove solvent completely using the rotary evaporator under reduced pressure at 40°C
(Water-bath temperature)
4. *Attach the test-tube to a rotary vacuum pump and dry the sample for about 5 min.
5. Leave the test-tube in a desiccator for 30 min and weigh the test-tube and contents
accurately.
* Drying can also be done in an electric air oven at 105°C for 30 min. However, the lipid may oxidise and hence
increase the weight of the dry sample by about 4 to 10%.
III CALCULATIONS
W1 Vt
Total lipid content (%) = X x 100
Ve Ws
where W1 = weight of dried lipid

Ws = weight of skin or meat used
Ve = volume of extract used
Vt = total volume of extract prepared
C- 3.1
D E T E R M IN A T IO N O F P H O S P H O L IP ID C O N T E N T
L O W L . K . & N G C .S .
P h o s p h o lip id s a re h y d ro ly z e d b y th e a c tio n o f p h o s p h o lip a s e . T h e a c tio n o f p h o s p h o li

p a se is u su a lly s tro n g e r th a n th a t o f lipase . T h e re fo re th e e x te n t o f h y d ro ly s is o f p h o s p h o lip id s
is a d o p te d as an in d e x o f lip id d e te rio ra tio n .
P h o s p h o lip id c o n te n t is o b ta in e d b y u sing c o lu m n c h ro m a to g ra p h y to s e p a ra te th e
trig ly c e rid e s (neutra l lip id s) fro m th e p h o s p h o lip id s . T h e p o la r p h o s p h o lip id s are a b s o rb e d by
th e s ilic ic a c id a n d is e lu te d b y th e m e th a n o l. T he neu tral lip id s w h ic h are n o t a b s o rb e d b y th e
s ilic ic a c id a re firs t e lu te d o u t b y th e c h lo ro fo rm .
I APPARATUS
G la ss c h ro m a to g ra p h c o lu m n (Ø :1 -2 cm ; le n g th : 30 cm ) w ith T e flo n ta p .
C o tto n w o o l
F ilter p a p e r (W h a tm a n N o. 1)
P re w e ig h e d , d ry e v a p o ra tin g fla s k (50 m l ca p a city)
A n a ly tic a l b a la n c e
D e s ic c a to r
R o ta ry e v a p o ra to r w ith w a te r b a th (28°C)
II REAG ENTS
a) S ilic ic a c id (M a llin c h ro d t, 100 m esh)
b) C e lite 545
c) M e th a n o l (a n a lytica l grade )
d) C h lo ro fo rm (a n a lytica l grade)
A. PR EP A R A TIO N O F T H E P A C K IN G M A TE R IA L
1. W ash th e s ilic ic a c id a n d c e lite 5 45 s e p a ra te ly w ith w a rm m e th a n o l fo r 5 to 10 m ins.
2. A llo w th e m a te ria l to s e ttle a n d d e c a n t th e w a s h in g so lu tio n .
3. R e p e a t s te p s 1. a n d 2. tw ic e .
4. W ash w ith w a rm a c e to n e tw ic e .
5. A ir d ry a t ro o m te m p e ra tu re o ve rn ig h t.
6. T he n o ve n d ry a t 120°C fo r 2 hou rs.
7. C o o l a n d m ix s ilic ic a c id a n d C e lite 5 45 in a ra tio 2:1.
B. PR EP AR A TIO N O F C O L U M N
1. W e ig h p a c k in g m a te ria l 1 0 -1 5 tim e s th a t o f th e lip id sa m p le w e ig h t.
2. S o a k c o tto n w o o l in c h lo ro fo rm a n d p a c k in to b o tto m o f co lu m n . E xclu d e as m u c h a ir as

p o s sib le .
C -4 .1
3. Place 2 layers of W hatm an No. 1 filter paper cut into size of columns.
4. M ix the packing material in chloroform and pour gently into column with the aid of a glass
rod.
5. Allow the packing m aterial to settle.
6. Place 2 layers of W hatm an No. 1 filter pap er on the packing material.
7. Drain column of excess chloroform leaving a 1 cm high column of chloroform.
III PROCEDURE
1. Dissolve 1 g sam ple lipid in pure chloroform, making a 5 to 1 0 % solution.
2. Introduce thin sam ple onto the column.
3. Drain off excess chloroform till solvent level is about 1 cm above the packing material.
4. Drain off with 2 5 0 ml chloroform and collect the neutral lipids in prew eighed evaporating
flask (elution speed: 3 drops per second).
5. Evaporate off the solvent using the rotary evaporator, dry up using a rotary vacuum pum p
for 5 mins, cool in desiccator for another 30 min and weigh th e neutral lipids.
6. Drain off with 100 ml m ethanol and collect the phospholipids in prew eighed evaporating
flask.
7. Evaporate off the solvent using the rotary evaporator, dry up using a rotary vacuum pum p
for 5 mins, cool in desiccator for 30 min and weigh the phospholipids.
IV C A L C U L A T IO N
(W eight of Neutral Lipid)

Neutral lipid (% ) = x 100
(W eight of PL + W t. of NL)
(W eight of Phospholipid)
Phospholipid (% ) = x 100
(W eight of PL + W t. of NL)
NL = Neutral lipid
PL = Phospholipid
The phospholipid content is expressed as the percentage of phospholipid over the total
lipid present per gram of sam ple lipid.
C -4 .2
DETERMINATION OF ACID VALUE
INTRODUCTION
The acid value is a measure of the extent to which the glycerides in the oil have been
hydrolysed by lipase action. The glycerides are also hydrolysed with water in the presence of air
and possibly bacteria. The decomposition is accelerated by heat and light.
As rancidity is usually accompanied by free fatty acid formation, determination of acid

value is often used as a general indication of the condition and edibility of oils.
The acid value is the number of milligrams of potassium hydroxide required to neutralize
the free fatty acids in 1.0 g of fat or oil.
I APPARATUS
Microburette (2 ml with 0.01 ml intervals)
Conical flasks (100 ml)
5 ml pipettes
II REAGENTS
a) 0.02N KOH in ethyl alcohol

Weigh 5.6 g of potassium hydroxide, dissolve in distilled water and make up to 100 ml with
distilled water (1N solution). Dilute 50 times with ethyl alcohol when required.
b) n-Hexane
c) 1% phenolphthalein or thymolphthalein in ethyl alcohol

Dissolve 1 g of either indicator in 100 ml of ethyl alcohol.
C-5.1
III PROCEDURE
1. Take 0.1 -0.3 g of fat sample or A ml of the extract containing 0.1 -0.3 g of fat in a 100 ml
Erlenmeyer flask.
2. Add [10 — A] ml of n-Hexane and 1-2 drops of indicator.
3. Titrate the solution against 0.02N KOH solution. The end point is reached when pink
(phenolphthalein) or blue (thymolphthalein) colour persists for 30 seconds.
4. Carry out a blank test using A ml of C-M Mixture instead of the extract.
IV CALCULATION
56.11 x 0.02 x (Vs — V b) x F
Acid value (mg/g) =
W
where Vs = titration volume of sample (ml);

Vb = titration volume of blank (ml);
W = weight of fat in the volume of extract usd (g);
F = factor of 0.02 KOH solution, where
5
F = : Vf is the volume of 0.02N KOH required to neutralize 5 ml of the
Vf 0.02N H2SO 4 solution.
56.11 = Molecular weight of KOH
0.02 = Concentration of KOH
C- 5.2
DETERMINATION OF FREE FATTY ACID (FFA)
LOW L. K. & NG C. S
INTRODUCTION
The FFA figure is usually calculated as oleic acid by dividing the acid value by 2. With most
oils the acidity begins to be noticeable to the palate when the FFA calculated as oleic acid is
about 0.5-1.5%.
When the FFA cannot be estimated in terms of oleic acid, it can be calculated from the
saponification value.
CALCULATION
1. Determination of Free Fatty Acid from Acid Value
mol. wt. of oleic acid 100

FFA (%) = acid value x X
mol. wt. of KOH 1000
282.27 1
= acid value x X
56.11 10
1
= acid value x
2
Determination of Free Fatty Acid from Acid Value And Saponification Value Expressed as
mg Number per 100 g Meat
acid value x total lipid

FFA (m g /100 g) = x 100
saponification value
* N.B. 2. Personal com m unication from Dr. Tsukuda.
REFERENCE
Pearson, D. (1976) In: The chemical analysis of foods (7th Ed.) Churchill Livingstone:493.
C -5.3
DETERMINATION OF SAPONIFICATION VALUE
INTRODUCTION
Saponification is the hydrolysis of esters. Oils and fats are the fatty acid esters of the
trihydroxy alcohol, glycerol. The saponification value of an oil is defined as the number of
milligrams of potassium hydroxide required to neutralise the fatty acids resulting from the
complete hydrolysis of 1 g of the sample. A soap is formed during saponification, for
example:
C3H5(C17H35COO)3 + 3KOH = C3H5(OH)3 + 3C17H35COOK
Stearin Glycerol Potassium stearate
The esters of the fatty acids of lower molecular weight require more alkali for
saponification, so the saponification value is inversely proportional to the mean of the molecular
weights of the fatty acids in the glycerides present.
As many oils have somewhat similar values, the saponification value is not, in general, so
useful for identification purposes. It is useful for detecting the presence of oil and fats which
contain a high proportion of lower fatty acids.
The fish lipid is extracted with C-M mixture and the solvent evaporated using the rotary
evaporator. About 0.2-0.5 g of lipid is used. The approximate sample sizes to be used for
each type of lipid is as follows:-
Sample size N/2 KOH solution

Type of lipid
(g) (ml)
fish lipid 0.2 - 0.5 10

animal fat 0.5 - 1.0 20
plant oil 0.5 - 1.0 20
wax 1.0 - 2.0 20
II APPARATUS
Bulb condensers
Erlenmeyers flasks (50-300 ml depending on sample size)
Water bath
Pipettes
Burette
III REAGENTS
a) 0.5N HCl standard solution
Use 1N HCl standard solution and dilute exactly two times.
C-6.1
b) 0.5N Ethanolic potassium hydroxide standard solution
Weigh 35 g of KOH, dissolve in 20 ml of water, then make up to 1000 ml with 95% (v/v)
ethanol or absolute alcohol.
c) Indicator
Phenolpthalein
Take 1 g of phenolpthalein and make up to 100 ml with 95% ethanol.
Methylene blue
Take 0.1 g of methylene blue and make up to 100 ml with water.
IV PROCEDURE
1. Take 0.2 to 0.5 g of lipid in a 50-100 ml Erlenmeyer flask.
2. Add 10 ml of 0.5N ethanolic potassium hydroxide solution and mix.
3. Heat at 80-85°C in a water bath for 30 min.
4. Cool to between 30-40°C liquid stato, then titrate with 0.5N HCl standard solution (Add 2-3
drops of indicator).
5. Carry out a blank test (without lipid).
V CALCULATION
28.05* x (A - B) x F
Saponification value (mg/g) =
S
where S = sample weight
A. = titration volume of blank (ml)
B = titration volume of sample (ml)
F = Factor of 0.5N HCl standard solution
* H alf o f m o lecu lar w e ig h t of K O H
REFERENCES
Jacobs, M.B. (1973). The chemical analysis of foods and food products (Reprint of 3rd Ed):
380-381.
Pearson, D. (1976). The chemical analysis of foods (7th Ed):491-492.
C-6.2
DETERMINATION OF PEROXIDE VALUE
INTRODUCTION
Unsaturated fish oils are particularly susceptible to oxidation, developing peroxides under
poor cold-storage or frozen storage conditions. Peroxides are the precursors of breakdown
products that cause rancid flavours in fat. The concentration of peroxides is indicative of
oxidation during the early stages of lipid deterioration. This index becomes less reliable during
the later stage of deterioration, because peroxide degradation increases.
The peroxide value (POV) is defined as the reactive oxygen contents expressed in terms of
milliequivalents (meq) of free iodine per kilogramme of fat. It is determined by titrating
iodine liberated from potassium iodide with sodium thiosulphate solution.
Oils with POV well below 10 meq/kg are considered fresh. A rancid taste begins to be
noticeable when the POV is between 20 and 40 meq/kg. In interpreting such figures, however,
it is necessary to take into account the particular oil or fat involved.
I APPARATUS
Evaporating flasks with stoppers (250 ml capacity)
Rotary evaporator with vacuum pump
Pipettes (1 ml, 5 ml, 10 ml, 20 ml)
Measuring cylinders (25 ml, 100 ml)
Stop watches
Microburette (2 ml)
Burette (50 ml)
Erlenmeyer flasks (100 ml, 200 ml) with stoppers
Balance with at least 0.1 g sensitivity
II REAGENTS
a) 0.01N Na2S2O3 solution
Dissolve 25 g of Na2S2O3.5H20 in freshly boiled distilled water and make up to 1000 ml.
Stand for 2-3 days. Add 10 ml of iso-amylalcohol as stabilizer. When required, dilute 10
times with freshly boiled distilled water. Keep in a dark brown bottle.
Standardization of the N2S2O3 Solution
1. Take 20 ml of 0.01N K2Cr2O7 solution in a 250 ml flask with stopper.
2. Add 10 ml of 10% Kl solution and 5 ml of 25% H2SO4.
3. Immediately stopper the flask and stand for 5 min in the dark.
4. Add 100 ml of distilled water and shake.
5. Titrate with 0.01N Na2S2O3 solution until yellow colour almost disappears.
6. Add 1 ml of 1.5% starch solution as indicator, and continue the titration until dark
blue colour disappears.
7. Carry out blank test by using 20 ml of distilled water instead of K2Cr2O7 solution.
C-7.1
8. Calculation:
20 x F'
F=
Vs – Vb
where F = factor of 0.01N Na2S3O3 solution

F' = factor of 0.01 N K2Cr2O7 solution
Vs = titration volume of sample (ml)
Vb = titration volume of blank (ml)
b) Chloroform-acetic acid mixture (2:3).

Mix CHCl3 and CH3COOH, 2:3 by volume. Flush with pure, dry nitrogen gas.
c) Saturated Kl solution
Dissolve 100 g Kl in 70 ml freshly boiled distilled water. Keep the solution with precipitated
crystals in a dark brown bottle.
d) 1.5% starch solution

Weigh 1.5 g of soluble starch in a beaker. Add 100 ml of distilled water. Heat and boil for
30 sec.
e) 0.01 N K2Cr2O7 standard solution

Weigh 4.9035 g of K2Cr2O7 which had been dried at 100-110°C for 3-4 hr. Dissolve it in
distilled water and make up to 1000 ml. When required, dilute 10 times with distilled water.
4.9035
Factor F' = ; where W is the actual weight of K2Cr2O7 used.
W
f) 10% (w/v) Kl solution

Dissolve 10 g of Kl in distilled water and make up to 100 ml
g) 25% H2SO4 solution

Mix 25 g (13.5 ml) of concentrated H2SO4 and 75 ml of distilled water.
Ill PROCEDURE
1. Take about 0.3 g of fat sample or A ml of the extract containing about 0.3 g of fat into a
250 ml flask with stopper.
2. Remove solvent using rotary evaporator under reduced pressure at 40°C (water-bath
temperature).
3. Add 10 ml of CHCl3-CH3COOH mixture and dissolve the fats by shaking.
4. Add 1 ml of saturated Kl solution.
5. Immediately stopper and stand in the dark for 5 min.
6. Add 20 ml of distilled water, then shake.
C-7.2
7. Titrate the liberated iodine with 0.01 N Na2S2O3 solution until light yellow colour. Add 1 ml
of 1.5% starch solution as indicator and titrate till colourless.
8. Carry out blank test in the same manner without fats.
IV CALCULATION
(Vs - Vb) x F x N x 1000 (Vs - Vb) x F x 1 x 1000

POV (meq per 1000 g) = =
W W x 100
(Vs – Vb) x F x 10
=
W
where Vs = titration volume of sample (ml);

Vb = titration volume of blank (ml);
F = factor of 0.01N Na2S2O3 solution;
W = weight of fat in volume of extract used (g);
N = normality of Na2S2O3 solution (in this case N/100)
0.5 x (Vs - Vb) x N x 1000

POV (millimoles per 1000 g) =
W
REFERENCES
Japanese Association of Oil Chemists: Standard methods of oil analysis in Japan, (1972)
2(4): 12-71.
Pearson, D. (1976). The chemical analysis of foods (7th Ed.): 494-495.
C-7.3
DETERMINATION OF THIOBARBITURIC ACID (TBA)
NUMBER
INTRODUCTION
In autoxidised lipids, most malonaldehyde does not appear in the free state but seems to
exist mainly in a weakly-bound state and is released when the system is heated with a mild
acid. The TBA test measures malonaldehyde in autoxidising systems. The basic reaction can
be represented as follows:-
Thio b arbitu ric M a io n a ld e h y d e Brilliant red p ro d u c t w ith

acid a b sorptio n m a x im u m a t 5 3 2 nm
It is a sensitive test and can be correlated with the development of off-odours and flavours.
It is especially well-suited for the detection of oxidative rancidity in lipids which are unsaturated
and contain 3 or more double bonds. The TBA number is defined as the number of
milligrammes of malonaldehyde per kilogramme of sample.
The results are expressed as malonaldehyde or 1,1,3,3,-tetra-ethoxypropane, which yields

malonaldehyde by acid hydrolysis.
I APPARATUS
Spectrophotometer (X = 532 nm)
Test tubes with screw caps
Hot water bath (boiling water)
Pipettes (3,5,10,25 ml)
Rotary evaporator with vacuum pump and water bath.
Vortex mixer
Test tube basket
Glass centrifuge tubes
Centrifuge
Source of N2 gas
C-8.1
II REAGENTS
a) TBA solution
Dissolve 1 g of TBA in 75 ml of 0.1 N NaOH. Dilute to 100 ml with distilled water (can be
kept for more than 1 month in refrigerator).
b) Tri-chloroacetic acid (TCA) solution

Mix 50 ml of 25% TCA solution, 30 ml of 0.6 N HCl and 420 ml of distilled water.
c) Antioxidant solution
Dissolve 0.3 g BHA (butylated hydroxyanisole) in 5.4 g propylene glycol. Dissolve 0.3 g of
BHT (butylated hydroxytoluene) in 4.0 g of warm Tween 20. Mix the two solutions.
d) Chloroform (Analytical grade)
III PROCEDURE
1. Take 0.2-0.4 g of fat sample or A ml of the extract containing 0.2-0.4 g of fat in a test tube
with screw caps.
2. Add 3 drops of antioxidant solution.
3. Remove the solvent using the rotary evaporator under reduced pressure at 35-40°C
(water-bath temperature).
4. Add 3 ml of TBA solution and 17 ml of TCA solution.
5. Flush N2 gas into the test tube and immediately stopper.
6. Heat at 100°C in a boiling water-bath for 30 min till the colour appears.
7. Cool to room temperature in tap water.
8. Add about 5 ml of chloroform and mix for a few seconds with a Vortex mixer.
9. Transfer about 15 ml of the colour solution to a GLASS centrifuging tube.
10. Centrifuge for 10 min at 3,000 rpm.
11. If the aqueous solution is not clear, centrifuge again at 10,000 rpm for 10 min.
12. Transfer a part of the clear aqueous solution and read absorbance at 532 nm.
13. Blank test should be carried out in the same manner without fats.
C-8.2
IV CALCULATION
Abs. x F x 0.2
TBA No. (mg malonaldehyde/kg fat) =
W
where Abs = absorbance at 532 nm

W = weight of fat in volume of extract (g)
F = factor = 46.
N .B . T h e a b s o rb a n c e o f a 1 g s a m p le (in 1 0 0 ml reagent) m ultiplied by th e fa c to r 4 6 is th e T B A n u m b er, o r th e

m illigram o f m a lo n a ld e h y d e p e r 1 0 0 0 g (1 kg) o f s a m p le (S in n h u ber et. al., 1 9 5 8 ). A s th e a m o u n t o f re a g e n t
u sed is only 2 0 m l, th e results m u s t b e m ultip lied by 0 .2 to give th e a b s o rb a n c e o f th e s a m p le in 1 0 0 ml re a g e n t
as s p ecified b y th e definition.
REFERENCES
Pearson, D. (1976). The chemical analysis of foods (7th Ed.):496-497.
Sinnhuber, R.O. and Yu, T.C. (1958). The 2-thio-barbituric acid method for the measurement of
rancidity in fishery product II. The quantitative determination of malonaldehyde. Food Tech.,
12:9.
Sinnhuber, R.O., and Yu, T.C. (1977). The 2-thio-barbituric acid reaction. An objective measure
of the oxidative deterioration occurring in fats and oils. Abura Kagaku, 26:259-267.
C-8.3
DETERMINATION OF METHYL ESTERS OF FATTY ACIDS BY
GAS CHROMATOGRAPHIC METHOD
INTRODUCTION
Methyl esters of fatty acids from fish and animal fats having 8-24 carbon atoms are
separated and determined by gas chromatography. This method is not applicable fo r epoxy,
oxidized, or polymerized fatty acids.
The fish oils used are first esterified by the boron triflouride method.
II APPARATUS
The following conditions are for use with flame ionization detector (FID).
a) Gas chromatograph (Shimadzu GC-9A).

With minimum dead space in injection system, which is maintained at 20-50°C higher than
column temperature. The column temperature should be maintained within ±1°C to at
least 220°C. If programmed heated, dual columns are used.
b) Columns
1.600 mm x 3 mm (i.d.) glass spiral columns.
Maximum aging temperature = 210°C.
c) Packing
Chromosorb W, (Acid-washed and silanized diatomaceous earth) mesh 60-80.
Coated with 5-20% diethylene glycol succinate (DEGS).
Condition column while disconnected from detector at 200°C with current o f nitrogen gas
at 60 m l/m in for 16-18 hours.
d) Microliter syringes
Maximum volume 10 ul, graduated to 0.1 ul (Hamilton 701-N).
e) Recorder (Chromatopac C-RIB, Shimadzu)

M ethod of recording : Therm al printer-plotter
Chart width : 21 cm
Chart speed : 0 -5 0 m m /m in
Pen speed : 0 .8 9 sec/full scale;

57 characters/sec.
Span : 1 mv (autom atic attenuation by tim e programming). With

attenuation switch to change range.
Integration sensitivity 1 uV. sec (= 1 digit)
Linearity ± 0 .1 % or better.
C- 9.1
III REAGENTS
a) Carrier gas
Purified grade nitrogen gas with oxygen < 4.0 ppm, moisture < 2.5 upm, hydrocarbons
< 1.0 ppm.
b) Other gas
Purified grade air with oxygen 21 ± 1%, moisture < 3.0 ppm, hydrocarbons < 5 .0 ppm.
Purified grade hydrogen with oxygen < 3 ppm, moisture hydrocarbons <1 ppm.
c) Reference standards
Known mixtures of methyl esters of fatty acids or methyl esters of oil of known
composition, preferably similar to that of material to be analyzed.
IV OPERATING CONDITIONS
a) Isothermal program
Column initial temperature = 180°C.
Column initial time = 0.0 min.
Column final temperature = 180°C.
Column final time = 500 mins.
Injection port temp. = 200°C.
Range = 102.
b) Gas flow rate and pressure

Hydrogen gas : 0.6 kg /cm 2
Purified air : 0.5 kg /c m 2
Nitrogen gas : 60 m l/m in.
c) Recorder conditions
Width : 5 sec
Slope : 300 uV/min
Drift : 0 uV/min
Min Area 10 count
T-DBL : 0 min
Lock : 1.3 min
Stop time : 1000 min
Attenuation 4 mV/full scale
Speed : 5 m m /m in
Method : 41
Sample weight : 100 (default value)
Internal standard weight : 1 (default value)
C -9.2
V PROCEDURE
1. With recorder showing stable baseline, inject 0.1-0.3ul 5-10% n-hexane solution of
methyl esters.
2. If trace com ponents are desired, the sample may be increased by < 1 0 times.
3. Pierce septum of inlet port and quickly discharge sample.
4. W ithdraw needle and note on chart small peak due to air or solvent, marking start
reference point.
5. Press ‘Start’ on both GC-9A and recorder.
6. Adjust sample size so that major peak is not attenuated > 8 times, preferably less.
7. Change setting of attenuator as necessary to keep peaks on chart paper. Mark attenuator
setting on chart.
VI IDENTIFICATION
1. Analyze reference standard mixtures under same operating conditions as for sample.
2. Measure retention tim e (S) for known esters by measuring the distances from start point.
3. Plot log S as a function of number of C atoms of acids. Under isothermal conditions,

graphs of straight chain esters of same degree of unsaturation should be straight lines,
approximately parallel.
4. Identify peaks from sample from these graphs, interpolating if necessary.
5. Avoid conditions which permit “ masked peaks” which are not sufficiently resolved.
N . B . E s t e r s a p p e a r i n o r d e r o f i n c r e a s i n g n u m b e r o f C a t o m s a n d o f i n c r e a s i n g u n s a t u r a t i o n f o r s a m e n u m b e r o f C
a t o m s . C 1 6 e s t e r i s a h e a d o f t h e C 1 8 e s t e r a n d C 1 8 M e e s t e r s a p p e a r i n o r d e r : s t e a r a t e ( 1 8 : 0 ) , o l e a t e ( 1 8 : 1 ) ,
l i n o l e a t e ( 1 8 : 2 ) , a n d l i n o l e n a t e ( 1 8 : 3 ) . C 2 0 s a t u r a t e d e s t e r ( a r a c h i d i c , 2 0 : 0 ) u s u a l l y a p p e a r s b e f o r e 1 8 : 3 e s t e r ,
b u t m a y b e r e v e r s e d o n s o m e c o l u m n s , o r p o s i t i o n s m a y c h a n g e w i t h c o l u m n u s e d .
VII CALCULATIONS
Method 41 of Chromatopac C-R1B is a normalization method. Use method of normalization,
which assumes all components of sample are represented on chromatogram, so that sum of
areas under peaks represent 100% of constituents (total elution). As the Chromatopac C-R1B
is equipped with integrator, the figures shown can be used directly for calculation. Report
results to following significant figures, with 1 figure beyond decimal point in all cases: 3 for
> 10% , 2 for 1-10% and 1 for < 1 % .
REFERENCES
Official methods of analysis of the Association of Official Analytical Chemists (13th Ed.), 1980
: 447-449.
Through personal communication with Mr Kinumaki and Dr Tsukuda.
C -9 .3
DETERMINATION OF THE DEGREE OF LIPID OXIDATION BY
GAS CHROMATOGRAPHY
INTRODUCTION
Fish oils, in general, consist predominantly of triglycerides and phospholipids, and minor
proportions of free fatty acids, vitamins, etc.
Fish oils contain approximately 15-40% (on the weight of total fatty acids) of saturated
fatty acids. The main saturated fatty acid is palmitic acid C16H32O2.
Polyenoic acids of the C16- 24 series occurs in fish oils. The acids of the C20 and C22 series
are the most abundant. An eicosapentaenoic acid, C20:5, and a docosahexaenoic acid, C22:6
occurs as a major component in most marine oils. It has been suggested that in the
docosahexaenoic acid the double bonds are either in the 4-5, 8-9, 12-13, 15-16, 18-19 and
21-22 or the 4-5, 8-9, 11-12, 14-15, 17-18 and 20-21 position.
Since both palmitic acid and docosahexaenoic acid are abundant in fish oils, we can use
them to measure the degree of lipid oxidation that has occurred during frozen storage.
The sample is prepared by the boron trifluoride method.
II PROCEDURE
The procedure is the same as that for the determination of Methyl esters of fatty acids
by gas chromatography.
III CALCULATION
The index of oxidation, I, is defined as:-
x’/y ’
I= 1–
x/y
where x’ = % of C22:6 of stored sample

y’ = % of C16:0 of stored sample
x = % of C 2 2 : 6 of fresh sample
y = % of C16:0 of fresh sample.
Hence, by measuring the ratio of C22:6 and C16:0 at the initial and subsequent stages, we
can use the index of oxidation as a measure of the degree of docosahexaenoic acid.
REFERENCE
By personal communication with Mr Kinumaki (1983).
C- 10.1
P R E P A R A T IO N O F M E T H Y L E S T E R S B Y B O R O N
T R IF L U O R ID E M E T H O D
Glycerides and phospholipids are saponified, and fatty acids are liberated and esterified in
presence of BF3 catalyst for further analysis by gas liquid chromatography (GLC).
This method is applicable to common animal and vegetable oils and fats, and fatty acids.
Unsaponifiables are not removed, and if present in large amounts, may interfere with
subsequent analyses.
This method is not suitable for preparation of methyl esters of fatty acids containing major
amounts of epoxy, hydroperoxy, formyl, oxo, cyclopropyl, and cyclo-propenyl groups, and
conjugated polyunsaturated and acetylenic compounds because of partial or complete
destruction of these groups.
I SAM PLE P R E P A R A T IO N
Precise weighing is not required. Sample size need be known only to determine size of
flask and amounts of reagents, according to following table:
Sample Flask 0.5N NaOH BF3 Reagent

(mg) (ml) (ml) (ml)
<50 10 1-2 2
50-75 10 2 3
75-100 10 3 4
100-250 50 4 5
250-500 50 6 7
500-750 100 8 9
750-1000 100 10 12
N .B . T ho u g h a 3 5 0 m g s a m p le is p re fe rre d , it m a y b e difficult to o b ta in this a m o u n t o f oil fro m low fa t s a m p le .

H e n c e , 5 0 -1 0 0 m g s a m p le c o u ld b e used.
II APPARATUS
Reaction flasks: 50 and 125 ml flasks with outer joints.

Condenser: Water-cooled, reflux, with 20-30 cm jacket and inner joint.
III REAGENTS
a) Boron trifluoride reagent — 125 g BF3/1 MeOH

Available commercially as boron trifluoride methanol complex with about 14% BF3. This
reagent is stable for 2 years.
(C au tio n : R e m o v e B F3 vap o u rs w ith e ffe c tiv e fu m e rem oval d e vice. A void c o n ta c t w ith skin, ey e s , and
respiratory tract).
C- 11.1
b) Methanolic sodium hydroxide solution (0.5N)
Dissolve 2 g NaOH in 100 ml MeOH containing <0.5% H2O. White precipitate of Na2CO3
forming on long standing may be ignored.
c) n-Hexane pure, as determined by GLC.
d) Nitrogen gas containing <5 mg oxygen/kg.
e) Methyl red solution — 0.1% in 60% ethyl alcohol.
IV PROCEDURE
1. Add sample (ca. 350 mg preferred for GLC) to flask and then add 0.5N methanolic NaOH
solution and anti bubbling stone.
2. Attach condenser, and reflux until fat globules disappear (usually 5-10 mins at
85°C ± 5°C).
3. Add BF3 solution from bulb or automatic pipette through condenser and continue boiling
for 2 min (at 90-100°C).
4. Add 2 ml n-hexane through condenser and boil for 1 min.
5. Remove heat, then condenser, and add several ml saturated NaCI solution.
6. Rotate flask gently several times.
7. Add additional saturated NaCI solution to float the n-hexane solution into neck of flask.
8. Transfer about 1 ml upper n-hexane solution into test tube and add small amount
anhydrous Na2SO4 to remove H2O. If necessary, dilute solution to concentration of 5-10%
for GLC.
N .B . BF3 is very to xic. W o rk in h ood. W a s h all g la s s w a re im m e d ia te ly a fte r use. If fa tty a c id s co ntain in g > 2 d o u b le
b o n d s a re p resen t, re m o v e air fro m M e O H an d flask by passing in s tream o f nitrogen g as fo r a fe w m in. M ethyl
esters should b e an a ly s e d as soon as possible. If n e c essary, n -h e x a n e solution m a y b e k e p t u n d e r N 2 in
refrigerator.
REFERENCE
Official methods of analysis of the Association of Official Analytical Chemists (13th Ed.),
1980: 447.
C -1 1.2
ANALYSIS OF ADDITIVES
D E T E C T IO N O F P O L Y P H O S P H A T E S
NG M .C .
Polyphosphates (food grade) are commonly used in the production of fish jelly products.
The addition of polyphosphates help to smoothen the ground fish paste and increase the gel
strength of the final fish jelly products. The commercial polyphosphate is a mixture of sodium
pyrophosphate and sodium tripolyphosphate (sodium triphosphate) of 1:1.
The principle of detection involves extracting the polyphosphates present in the sample
with trichloroacetic acid, separating the phosphates by thin layer chromatography (TLC) and
finally detecting the phosphates by spraying with colour reagent.
This method is also applicable to meat and meat products.
I P R E P A R A T IO N OF SAM PLE S O L U T IO N
1. Mix well 50 g minced sample with 15 ml warm water using a spatula.
2. Add 10 g trichloroacetic acid and mix.
3. Store in the refrigerator for one hour to allow separation.
4. Filter the separated solution.
5. Collect the clear solution for chromatographic separation.
N.B. 1. If the filtrate is turbid, add an equal volume of diethylether and shake. Remove the ether layer with small
pipette and add an equal volume of 95% ethanol to the water phase. Shake for a minute. Allow the
mixture to stand for a few minutes before filter.
2. Use the sample solution on the day of preparation. Store it chilled if the chromatography analysis cannot
be done immediately.
II REAGENTS
a) Preparation of TLC plates

1. Weigh 15 g of cellulose powder, Whatman thin layer chromedia CC41, into a beaker.
2. Add 30 ml distilled water and mix well with glass rod.
3. Apply this slurry onto glass plates (20 x 20 cm) with the spreading device to obtain a
layer of 0.25 mm in thickness.
4. Air-dry the plates undisturbed for 60 min at room temperature.
5. Heat them finally for 10 min at 100°C.
6. Store the plates in a desiccator.
D-1.1
b) Preparation of developing solvent of TLC
Isopropyl alcohol, 140 ml.
Trichloroacetic acid (TCA), 40 ml of 13.5% solution.
Ammonia, 0.6 ml (SG 0.91).
Mix these solutions. If the solvent is not to be used on the same day of preparation, keep it
in a tightly closed bottle.
c) Preparation of spray reagents

Spray reagent I
7.5% ammonium molybdate solution
Concentrated nitric acid (analytical grade)
Mix equal volumes (1:1). Prepare the reagent on the day of use.
Spray reagent II
195 ml of 15% sodium metabisulphite solution
5 ml of 20% sodium sulphite
0.5 g 1-amino-2-naphthol-4-sulphonic acid
Mix and store the reagent in a closed brown bottle in the refrigerator.
Spraying with reagent II is not an absolute necessity. However the intense blue spots
produced by these reagents improve the detection considerably.
d) Preparation of polyphosphate standards

Sodium dihydrogen orthophosphate monohydrate, NaH2PO4.H20
Tetrasodium diphosphate decahydrate, (sodium pyrophosphate) Na4P2O7.10H2O
Pentasodium triphosphate, (sodium tripolyphosphate) Na5P3O10
Sodium hexametaphosphate (NaP03)6, Gramham’s salt
Dissolve 200 to 300 mg of each of the standards in 100 ml of distilled water. These
standard solutions can be kept at 40°C for 4 weeks.
III INSTRUMENTS/APPARATUS
Oven (Temp 30-200°C)
Glass chamber tank with cover
TLC plates (20 x 20 cm)
Glass tips
IV CHROMATOGRAPHIC SEPARATION OF POLYPHOSPHATES

1. Fill a paper-lined developing chamber with the developing solvent up to a layer of 0.5 to 1
cm over the bottom. Close the chamber immediately with a tightly fitting lid.
2. Allow to stand for at least 30 min at ambient temperature in order to saturate the chamber
atmosphere with the vapour of the developing solvent. This system should be protected
from sunlight and draught.
3. Apply 5 μl of the sample solution on to the TLC plate at about 2 cm from the bottom end of
the plate. Keep the spots small by applying 1 μl at a time. Use a cold air stream for drying.
D -1 .2
N.B. Hot air should be avoided because of danger of hydrolysis of polyphosphates.
4. In the same way, apply 5 μl of the standard solutions on the plate at an interval of 1.5-2cm,
but at exactly the same distance from the bottom end of the plate.
5. Remove the lid from the chamber and quickly but carefully place and dip the spotted plate
in the developing solvent in the chamber. Replace the lid immediately.
6. Develop the plate until the solvent front has ascended about 10 cm.
7. Remove the developed plate from the chamber, mark the position of the solvent front with
pencil, and allow to dry at ambient temperature for 30 min or, alternatively, in a stream of
air.
8. Place the plate under a fume hood and spray the plate lightly but uniformly with
spray reagent I.
9. Air-dry the plate under a fume hood. Subsequently heat for 30 min at 100°C in the oven in
order to remove the last traces of nitric acid and decompose polyphosphate.
10. Remove the plate from the oven and verify the absence of the pungent smell of nitric acid.
Yellow spots will slightly appear in the presence of phosphate.
11. Allow the plate to cool to room temperature and then replace it under the fume hood.
Spray the plate lightly but uniformly with spray reagent II. Blue spots will appear
immediately on phosphate areas.
12. Measure the migrating distance from the spotting position to the center of the phosphate
spot (A) and also to the solvent front (B).
13. Calculate the ratio A/B (Rf value).
V IN T E R P R E T A T I O N O F RESULTS
Compare the migrating distance of the phosphate spots from the sample with those of the
standard solution. The Rf values of some phosphates are:-
Orthophosphate 0.80 - 0.90

Diphosphate (Pyrophosphate) 0.40 - 0.45
Triphosphate 0.20 - 0.23
Hexametapolyphosphate 0.00
These values are changeable according to developing conditions. It is advisable to analyse

sample solution together with the standard solution.
REFERENCE
International Standard Orgn. ISO/TC 34/Sc 6/WG 2. The Netherlands.
D -1 .3
DETERMINATION OF MONOSODIUM L-GLUTAMATE (MSG)
CONTENT IN FISH JELLY PRODUCTS
NG M .C.
INTRODUCTION
Monosodium L-glutamate is usually used as a taste enhancer in the production of fish jelly
products.
The presence of MSG present in fish jelly products can be determined by enzymatic
reaction. In the presence of the enzyme, glutamate dehydrogenase (GIDH), the L-glutamic acid
present is deaminated oxidatively by nicotinamide-adenine dinucleotide (NAD+) to α-
ketoglutarate (see reaction 1). In the reaction catalyzed by diaphorase, the NADH formed
converts iodonitro tetrazolium chloride (INT) to a formazan which is measured in the visible
range at 492 nm (see reaction 2).
GIDH
(1) L-Glutamate + NAD+ + H2O → α-ketoglutarate + NADH + NH4+
diaphorase,
(2) NADH + INT + H + → NAD+ + formazan
The equilibrium of reaction (1) lies far on the side of glutamate. By trapping the NADH
formed with INT (2), the equilibrium is displaced in favour of α-ketoglutarate.
Collect fish jelly products sample (≤ 100 g) and pass 2-3 times through food mincer, or
chop very finely and mix thoroughly.
II REAGENTS
a) Preparation of standard L-glutamic acid solution
1. Dissolve 100 mg L-glutamic acid with 25 ml distilled water.
2. Adjust to pH 7.0 with 2N KOH.
4. Pipette 10 ml solution into a volumetric flask.
This is the standard solution which contains 40 mg L-glutamic acid/litre.
b) Dilution of standard L-glutamic acid (40 mg/litre)

0.2 ml of the above solution was pipetted into 1.8 ml of distilled water. This contains 8.0 ug
L-glutamic acid.
c) 1 M perchloric acid, HClO4

Dissolve 143.51 g perchloric acid in 1 litre distilled water.
D-2.1
d) 2N KOH
Dissolve 11.2 g KOH in distilled water and make up to 100 ml in a volumetric flask.
e) Treated sand
f) Enzyme solution (1 set contains 4 enzyme solutions named solution 1, 2, 3 and 4)
N.B. This enzyme solution can be purchased from:

Food Analysis
Boehringer
Mannheim GmbH
Manneheim, WEST GERMANY
III PROCEDURE
A. PREPARATION OF L-GLUTAMIC ACID SAMPLE SOLUTION
* Supernatant sample is to be diluted if too concentrated.
D-2.2
B. PREPARATION OF SUPERNATANT SAMPLE IN ENZYME SOLUTION
Pippette the enzyme soln and sample soln into test-tubes according to the following table
(duplicate) and mix. Add soln 4 and mix again. Stand for 30 min at 25°C water bath. Read the
optical densities of the soln at 492 nm.
Blank Standard Sample

(ml) (ml) (ml).
Enzyme Soln. 1 0.60 0.60 0.60
Enzyme Soln. 2 0.20 0.20 0.20
Enzyme Soln. 3 0.20 0.20 0.20
Distilled Water 2.00 — —
Std. Soln. — 2.00 —
Sample Soln. — — 2.00
Soln. 4 0.03 0.03 0.03
IV CALCULATION
To calculate the L-glutamic acid in fishball
O.D.sample = O.D.sample - O.D.blank
O.D. sample x 8x 10–3 x D x 50 X [40 + 5M] X 100

L-glutamic acid (mg/100 g) =
O.D. standard 2 25 W
where O.D.sample = optical density of sample

where O.D.standard = optical density of standard
8 x 10-3 = standard solution used in mg L-glutamic acid
M = moisture of sample
D = Dilution ratio
W = Wt. of sample (5 g)
50 Make-up volume
2 Sample of volume of reaction
[40 + 5 m] = Volume of perchloric acid solution used.
REFERENCE
Colormetric method for the determination of L-glutamic acid in foodstuffs. Cat. No. 139092.
Available from Boehringer Mannheim, GMBH. West Germany.
D-2.3
DETERMINATION OF SUGAR (SUCROSE)
BY SOMOGYI’S METHOD
NG M. C.
INTRODUCTION
Sugar is widely used in the manufacturing of food as taste and flavour enhancer. It is also
capable of inhibiting, retarding or arresting the process of fermentation, acidification or any
other decomposition of food. Thus sugar is also used as a preservative.
The sugar extracted from the sample is converted into glucose with diluted HCI. The
glucose content is determined by Somogyi’s method. The content of sugar is then back
calculated from glucose content. The recovery of sugar was found to be 91% and the
reproducibility was satisfactory.
Take a representative sample of the product, pass it through the mincer, transfer into a
labelled polyethylene bag and keep it chill.
II REAGENTS
a) Somogyi solution A
b) Somogyi solution B
D-3.1
c) 0.1N HCl
Dilute 10 ml 1N HCI in 100 ml volumetric flask.
d) 0.1N NaOH
Weigh 1 g NaOH, dissolve in distilled water and make up to 250 ml volumetric flask.
e) 2N H2SO4
Conc. H2SO4 60 ml dilute to 1 litre.
f) Starch indicator
Weigh 1 g soluble starch and 0.1 g salicylic acid, dissolve both in 99 ml distilled water. Boil
to dissolve the starch.
g) Dried KIO3
Weigh about 2 g of KIO3, dried in the oven at 120°C for 1 hr.
h) 2.5% Kl
Weigh 2.5 g Kl, dissolve in 97.5 ml of distilled water.
i) 0.05N Na2S2O3 solution

Sodium thiosulphate Na2S2O3.5H2O, 13 g.
-------------------- Na2CO3 0.2 g
Make up to 1 litre with decarbonated H2O
j) 0.005N Na2S2O3
Dilute 100 ml of 0.05N Na2S2O3 to 1 litre.
DETERMINATION OF FACTOR (F) OF 0.05N Na2S2O3
Weigh about 1.5 g dried KIO3 accurately
Make up to 500 ml with H2O in volumetric flask
Pipette 10 solution Blank

Pipette H2O, 10 ml
Add 2.5% Kl, 20 ml —
Add 2N H2SO4, 20 ml
Titrate with 0.05N Na2S2O3 with starch indicator

10 1 1
Factor, F = wt. of KIO3 x X X
500 0.0017835 ( B – A)
0.0017835: conversion factor of 1 ml 0.05N Na2S2O3 to KIO3 (g)

A: titration volume of KIO3 solution (ml)
B: titration volume of blank (ml)
D—3.2
Ill PROCEDURE
Minced fishball (S = 25 g)
----------------- 200 ml 60-70°Cwater
Homogenise
Centrifuge at 2000 rpm 5 min
— ------------- discard ppte
Supernatant
Make up to 250 ml with H2O
----------------- pipette 50 ml
----------------- add 0.1N HCI 15 ml using a measuring cylinder
Put in boiling water bath for 30 min
Cool down in ice
Neutralise with 0.1N NaOH using pH meter
Make up to 100 ml with H2O
Blank
Pipette 5 ml Pipette 5 ml distilled H2O
------------------Add Somogyi solution A 5 ml:---------------------

Mix well by swirling and place in
boiling water bath for 15 min
with aluminium foil cap.
------------------Cool down in ice, don’t stir.------------------------
-------------- — Add Somogyi solution B 2 ml.--------------------

Don’t agitate.
------------------Add 2N H2SO4 3 ml---------------------------------

using bulb pipette.
------------------ Mix thoroughly and stand---------------------------

for 2 min.
------------------ Titrate with 0.005N Na2S2O3------------------------

using starch indicator.
D-3.3
IV CALCULATION
100 250 1
Sucrose (%) = 0.0001449 (B - A) F x X x 0.95 x x 100
5 50 S
1
= 13.7655 (B - A) F x
S
where 0.0001449 : 1 ml 0.005N Na2S2O3 = 0.0001449 g glucose
A : Sample titration volume (ml)
B : Blank titration volume (ml)
F : Correction factor of Na2S2O3
S : Sample weight
0.95 : Conversion factor of glucose to sucrose
REFERENCES
David Pearson. The chemical analysis of food. 7th Ed: 128.
Official methods of analysis of the Association of Official Analytical Chemists. 13th Ed. 1980.
1980: 226, 14.114(d)
877, 50.037, 50.038
515, 31.052, 31.053
D—3.4
DETERMINATION OF STARCH
NG M. C.
INTRODUCTION
Starch is commonly used in the production of fish jelly products. Its main functions are:-
(1) as an extender to increase the bulk of production
(2) as a binding agent
Starch is readily convertible into glucose by hydrolysis either by an enzyme such as

diastase or by heating with an acid and its estimation usually depends upon this reason.
The hydrolysed glucose is determined by Somogyi method (D-3). The content of starch in
the sample is then back calculated from the content of the glucose.
For the confirmation of presence of starch in fish jelly product, the sample is first heated
with water. The starch granules will swell up and burst at about 70°C, resulting in a sticky feel.
When iodine solution is added, a characteristic blue colour is developed, due to starch iodide,
which is decomposed on heating, but is reformed on cooling.
Collect fish jelly product sample (≤ 100 g) and pass 2-3 times through food mincer, or
II REAGENTS
a) 8% potassium hydroxide (KOH) in alcohol.
Dissolve 8 g KOH in 4 ml of distilled water completely and mix with 96 ml absolute alcohol.
(Potassium hydroxide must be dissolved in water first as it is insoluble in ethanol).
b) 50% ethanol.
c) 2.5% hydrochloric acid (HCI)

14 ml conc. HCI in 186 ml distilled water (Conc. HCI is 35%).
d) 15% Sodium hydroxide (NaOH)

15 g NaOH (Technical grade) dissolve in 85 ml distilled water.
D-4.1
e) Somogyi solution A
Keep at room temperature.
f) Somogyi solution B
Keep at room temperature.
g) 2N H2SO4
Cone. H2SO4 (60 ml) dilute to 1 litre with distilled water. (Conc. H2SO4, 36N, is 95-97 wt
%).
h) Starch indicator
Dissolve 1 g soluble starch and 0.1 g salicylic acid in 99 ml distilled water. Boil to dissolve
the suspension.
i) Dried KlO3
Weigh about 2 g KlO3 and dry in oven at 120°C for 1 hour.
j) 2.5% Kl
Dissolve 2.5 g Kl in distilled water and make up to 100 ml.
D-4.2
k) 0.05N Na2S2O3 solution
Sodium thiosulphate Na2S2O3.5H2O 13g
------Na2CO3 0.3 g
Make up to 1 litre with decarbonated distilled water.
Determination of factor, F, of 0.05N Na2S2O3

Weigh 1.5 g dried KlO3 accurately and dissolve in 500 ml distilled water in volumetric flask.
To 10 ml of KlO3 solution and 10 ml distilled water (BLANK) each add 2.5% Kl (20 ml) and
2N H2SO4 (20 ml).
Titrate with 0.05N Na2S2O3 using starch indicator.
10 1 1
Factor, F = Wt. of KIO3 x X X
500 0.0017835 (B – A)
where 0.0017835: conversion factor of 1 ml 0.05N Na2S2O3 to KlO3

A: titration volume of KI03 solution (ml)
B: titration volume of blank (ml)
III PROCEDURE
Minced fish ball (10 g)
----------Put in centrifuge tube
---------- 8% KOH-alcohol (50 ml)
Heat at 90-95°C with condenser till starch precipitant occurs (usually 30-40 min.)
Cool down
Centrifuge at 2000 rpm/5 min
----------discard supernatant
ppte
-------- —wash with 50% alcohol (25 ml)
Centrifuge
---------- discard supernatant
ppte
----------transfer ppte into 300 ml Erlenmeyer flask with 200 ml of 2.5% HCI
----------boil for 2½ hours in water bath with condenser, cool down
---------- adjust to pH 6.5-7.0 with 15% NaOH using pH meter

V
D-4.3
V
-----------make up to 500 ml with distilled H2O
-----------pipette 10 ml into 100 ml Erlenmeyer flask
Blank
water 10 ml
-----------add Somogyi solution A (20 ml)---------------------------------
-----------place in boiling water bath----------------------------------------

with aluminium foil cap for 25 min.
-----------cool down immediately in ice water----------------------------
-----------add Somogyi solution B (10 m l)---------------------------------
-----------2N H2SO4 (10 ml)----------------------------------------------------
(must be fast while adding, use pasteur pipette)
----------------------------------- mix well-------------------------------------------
------------------------------ stand for 2 min-------------------------------------
Titrate with 0.05N Na2S2O3 using starch indicator
(Note: colour changes to light blue).
The blank test with distilled water (10 ml) should be carried out simultaneously with the
supernatant sample.
IV CALCULATION
500 1
Starch (%) = 0.001499 (B - A) F x x 0.9 x x 100
10 S
A = titration volume of sample (ml)

B = titration volume of blank (ml)
F = Factor of 0.05N Na2S2O3
0.001449= Conversion factor of 0.05N Na2S2O3 (ml) to glucose (g)
0.9 = Conversion factor of glucose to starch.
S = Weight of sample (g).
REFERENCE
Official methods of analysis of the Association of Official Analytical Chemists. 13th Ed. 1980.
24.057:383.
D-4.4
DETERMINATION OF SALT
NG M .C .
INTRODUCTION
Sodium chloride (Food grade) is an important additive for the production of fish jelly
products. Its main function is to extract the salt soluble protein to give the gel strength of the
final product.
The amount of sodium chloride present in such products can be determined by titrating
the extract containing the chloride ion with silver nitrate, AgNO3. Potassium chromate (K2CrO4)
is used as the indicator and the end point is indicated by the change in colour from yellow to
reddish brown.
I PREPARATION
Collect fish jelly products sample (≤ 100 g) and pass 2-3 times through food mincer, or
II REAGENTS
All reagents should be of GR grade or AR grade:-
a) 0.1N silver nitrate (AgNO3) solution

Dissolve 17 g of AgNO3 in distilled water and make up to 1 litre in volumetric flask. Keep it
in a brown colour glass bottle in the dark.
b) Potassium chromate indicator, K2CrO4

Dissolve 5 g K2CrO4 in distilled water and dilute to 100 ml.
III PROCEDURE
1. Weigh accurately 25 g sample into a 400 ml beaker.
2. Add 200 ml hot boiled water and stir for 60 mins.
3. Filter through the glass wool. Collect the filtrate in a 250 ml volumetric flask. Make up to
the volume and shake well.
4. Transfer 10 ml filtrate with bulb pipette into 100 ml conical flask. Add 50 ml distilled water
using the measuring cylinder and 1 ml K2CrO4 indicator.
5. Titrate with 0.1N AgNO3 (S ml). At the end point, the colour changes from yellow to
brownish red.
6. Carry out a blank determination using 60 ml distilled water and 1 ml K2CrO4 indicator (B
ml).
D-5.1
IV CALCULATION
250 ml
Salt (%) = x (S - B) x F x 100
10 ml x 25 g
where S = Titration volume of sample (ml)

B = Titration volume of blank (ml)
F = Conversion factor of 1 ml 0.1N AgNO3 to 0.005844 g NaCl
REFERENCES
David Pearson. The chemical analysis of food. 7th Ed: 519.
Official method of analysis of the Association of Official Analytical Chemists 13th Ed. 1980:289,
18.034.
D-5.2
SEMI-QUANTITATIVE ANALYSIS OF BORIC ACID AND
BORATES IN MEAT AND MEAT PRODUCTS
NG M. C.
INTRODUCTION
Boric acid and borates were commonly used as a preservatives. It. acts as an
anti-microbiological agent. However these preservatives are not permitted in the fishery
products.
In the presence of boric acid (H3BO3) or sodium borate (Na2B4O7) the turmeric test paper
turns methyl red. This can be further confirmed by addition of NH4OH which changes test
paper to dark blue-green, but restored to red by acid.
In the semi-quantitative analysis, the amount of boric acid or borates detected is

compared with the degree of redness on the turmeric test paper prepared from a range of
standard boric acid (0-1%).
This method is applicable to meat and meat products.
Collect meat sample (≤ 100 g) and pass 2-3 times through food mincer, or chop very finely
and mix thoroughly.
II REAGENTS
a) Hydrochloric acid, conc.
b) 80% Ethanol
c) Preparation of turmeric paper

Add 100 ml 80% ethanol to 1.5-2.0 g turmeric powder in 250 ml Erlenmeyer flask. Shake 5
min and filter. Dip sheets of Whatman No. 1 paper into the clean filtrate in flat-bottom dish
(eg petric dish). Hang paper to dry. After 1 hour, cut into 6 x 1 cm strips and store in tightly
stoppered container protected from light.
d) Preparation of reference standards

1. Dissolve 1.000 g H3BO3 in distilled water and dilute to 100 ml with distilled water.
2. Transfer 0.00, 0.10, 0.20, 0.50, 0.75, 1.00, 2.50 and 5.00 ml of the above H3BO3
solution to 15 ml test tubes.
3. Dilute to 10 ml with distilled water and add 0.7 ml HCl to prepare the reference
standard.
4. Keep tubes tightly stoppered to prevent evaporation.
These references standard solutions represent 0.00, 0.02, 0.04, 0.10, 0.15, 0.20, 0.50 and
1.00 % H3BO3 in meat (based on 25 g sample extracted with 50 ml distilled water and 10
ml aliquot used for test). The standard solutions may be stored in pyrex test tubes for more
than 6 months.
D-6.1
III APPARATUS
Erlenmeyer flask (125 ml)
Glass rod for stirring
Watch glass
Bunsen burner
Test tubes
Petri dish
Forceps
Scissor and string
IV PROCEDURE
1. Disperse 25 g of ground meat in 50 ml distilled water in 125 ml Erlenmeyer flask, using
flat-end stirring rod. Cover with watch glass.
2. Bring to boil over medium flame with agitation. Do not over-heat.
3. Cool in ice bath until fat solidified (30 min).
4. Filter through pledget of glass wool.
5. Transfer 10 ml filtrate to 15 ml test tube, add 0.7 ml HCI, stopper, and mix.
6. Mark identification on end of piece of turmeric paper and dip unmarked end into unknown
solution to ½ the length of paper.
7. Quickly remove moistened paper and place on sheet of white filter paper. Flat-tipped
forceps are useful in handling paper.
8. Place freshly prepared standard strips of test paper (made by dipping turmeric papers in
similar manner into series of standard solutions) alongside sample turmeric strips.
9. After more than 1 hour (but < 2 hour) at room temperature, strips are dry enough for
comparison. Good natural light is preferred.
V INTERPRETATION OF RESULTS
Place standard strips ca 1 cm apart on white filter paper background and bring
“ unknown” sample strips between adjacent standard strips for close colour matching.
If colour intensity is beyond range of standards, repeat test with dilution of meat filtrate (eg
5 ml filtrate. 5 ml distilled water, 0.7 ml HCl, and multiply final reading by 2). Use freshly
prepared set of standards with each series of samples tested.
REFERENCE
Official methods of analysis of the Association of Official Analytical Chemists. 13th Ed.
1980. 20.33:328.
D-6.2
MICROBIOLOGICAL PROCEDURE
HANDLING OF FOOD SAMPLES
LIM P.Y.
I COLLECTION, TRANSPORT AND STORAGE OF SAMPLES

a) Samples shall be transported to the laboratory as soon as possible after sampling, and
shall reach the laboratory within 24 hours of sampling.
b) Samples shipped frozen should be frozen when received by the laboratory. Fresh
perishable samples should register a temperature from 0°C to 4°C.
c) Ideally, samples should be examined immediately upon receipt by the laboratory.

Practically however, initiation of analysis may have to be postponed. Store frozen samples
at -2 0 °C until they are to be examined. Fresh or refrigerated products are stored between
0° and 4°C for not longer than 24 hours. Store non-perishable, canned, or low-moisture
food at room temperature until ready for analysis.
II CONDITION OF SAMPLES CONTAINER

Checking sampling containers for gross physical defects. Carefully inspect plastic bags
and bottles for tears, pinholes and puncture marks. Any cross-contamination resulting
from one or more of the above defects would invalidate the sample. Samples should be
adequately sealed and labelled.
III THAWING
When necessary to thaw the sample, use aseptic technique (e.g. in laminar flow chamber)
throughout the handling of the product. If the sample is frozen, thaw it in the original
container or in the container in which it was received in the laboratory. Whenever possible,
avoid transferring the sample to a second container for thawing. If the sample can be
easily handled without thawing, e.g. ice cream, proceed directly to the next step. If the
frozen sample must be thawed, do it in a manner that minimizes destruction or
proliferation of the sample microflora. Normally, the sample can be thawed at 2-5°C within
18 hours. If rapid thawing is desired, thaw the sample at less than 45°C for not more than
15 mins. When thawing a sample at elevated temperatures, agitate the sample frequently,
or preferably, continuously. Such rapid thawing is best carried out in a controlled
temperature water-bath.
E—1.1
AEROBIC PLATE COUNT
LIM P.Y.
INTRODUCTION
The aerobic plate count provides an estimate of the number of viable micro-organisms in
food according to the medium used and the time and temperature of incubation. The spread
plate method described below is based on the assumption that each viable cell will form a
colony, thus it is important that:-
— the sample is adequately dispersed

— the cells do not lose their viability
— the cells do not multiply during the preparation of the dilutions.
The material under investigation is diluted in known volumes of sterile diluent to provide a
set of serial dilutions of the microbial population so that an aliquot at some step in the series
provides 30 to 300 colonies when plated on a nutrient medium. (It is this count that will give the
most accurate colony count.)
I CULTURE MEDIA*
Plate count agar (PCA) or Standard Methods agar
Butterfield’s buffered phosphate diluent.
* R efer to A p p e n d ix B fo r m e th o d s of m e d ia p re p aration .
II APPARATUS
‘Waring’ blender & flasks Autoclave
Pipettes Incubator
Scissors & forceps Water-bath
Alcohol lamps Weighing balance
Alcohol (70% v/v) swabs Laminar flow chamber
Bent glass spreader
III SAMPLING PROCEDURE

Randomly pick 150-200 g of sample. Aseptically slice each piece of the sample in half and
keep the half-cut portions in a sterile polyethylene bag or container. Store in refrigerator
(5°C) to maintain its integrity.
IV SAMPLE PREPARATION
1. Weigh about 50 g of the above sample and put them into a ‘Waring’ blender flask. Add in
450 ml of sterile Butterfield’s buffered phosphate diluent. Blend for 1 min at low speed.
2. Transfer 5 ml of this suspension into 45 ml of phosphate diluent to give a dilution of 10- 1.

Prepare further dilutions by mixing 1 ml of the well mixed diluted sample solution with 9 ml
of phosphate diluent.
E-2.1
V PROCEDURES
1. Select the appropriate dilutions and for every dilution, inoculate 0.1 ml aliquots to each of
two PCA plates.
2. Spread the inoculum gently and evenly over the surfaces of the agar plates with a sterile
bent glass spreader.
3. Allow the plates to stand until the inoculum has been absorbed completely, which should
be within 15 mins after the spreading.
4. Invert the plates and incubate at 35°C for 48 hrs or at any suitable temperature and period.
5. Count those plates which have between 30-300 colonies.
6. The Aerobic plate count for the sample is calculated as below:-
Average number of colonies on the plate x Dilution factor x 10
Calculate to per gram of the sample. (See below).
VI CALCULATION OF AEROBIC PLATE COUNT (Spread Plate Method)

450 + W 1 En + En’
APC = X x 10 x organisms/g
W d P + (0.1 )q
where d:lowest dilution

En : total count of 2 plates atlowest dilution
En’ : total count of 2 plates athighest dilution
p : number of plates at lowest dilution
q : number of plates at highest dilution
W : weight of sample
VII BACTERIOLOGICAL LIMITS OF APC FOR FISH/FISHERY PRODUCTS (COOKED &

RAW)
Total plate count at 35°C for 48 hrs for
cooked products : 1 x 106 orgs/g
raw products : 2.5 x 106 orgs/g
REFERENCES
A. Hazzzard. (1985) ASEAN Training Course in Fish Quality Control. Training Course organised
by HAWKAID, Hawkesbury Agricultural College Research and Development Co. Ltd.
Chapter: Fish quality control microbiology. Section 4:63-65
Veterinary Public Health Laboratory, Primary Production Department unpublished manual.
E-2.2
A flow diagram of the procedure for Aerobic Plate Count (APC) is included as the following
figure.
FLOW DIAGRAM OF THE PROCEDURE FOR AEROBIC PLATE COUNT (APC)
Sample
V
10–1, 10–2, 10–3 dilutions . . . .
0.1 ml of each dilution

spread plate (in duplicate)
35°C/48 hrs*
Counting of plates with 30 - 300 colonies
Calculation of APC to orgs/g
* o r a n y s u ita b le te m p e ra tu re and period o f incubation.
E-2.3
COLIFORMS AND ESCHERICHIA COLI
LIM P. Y.
INTRODUCTION
Coliforms are Gram-negative, non-sporing, facultatively anaerobic rods which ferment

lactose, producing acid and gas within 48 hrs and they belong to the family Enterobacteriaceae.
The coliform group includes several genera, some of which are of intestinal origin (Escherichia)
while others are associated with plant and soil material (Enterobacter). Thus it is actually a
misconception to consider the coliform group as simply an indicator of faecal pollution.
However, generally speaking, it is the count of E. coli that is a more reliable indicator of
faecal contamination. Its presence indicates recent faecal contamination as it generally does
not survive for long in environments other than the intestine.
Faecal coliforms are a group of coliforms capable of fermenting lactose to produce acid
and gas at both 37°C and 44.5 ± 0.5°C in 48 hrs and generally contain a high proportion of
E. coli. As a significant number of non-faecal coliforms can give a positive faecal coliform test,
the test can be made more specific for E. coli by testing for the production of indole at
44.5 ± 0.5°C.
I CULTURE MEDIA*
Brilliant green bile broth (BGB)
Butterfield’s buffered phosphate diluent
Eosin methylene blue agar (EMB)
Koser citrate medium
Lauryl sulphate tryptose broth (LST)
MRVP medium
SIM medium
Simmons citrate agar
Nutrient broth
* R efe r to A p p e n d ix B fo r m e th o d s o f m e d ia p re p aration .
II CHEMICAL REAGENTS#
a) Kovac’s reagent
b) Methyl red solution
c) α-naphthol solution (5% w/v)
d) KOH solution (40% w/v)
# R efer to A p p e n d ix D fo r m e th o d s o f re a g e n t p re p aration .
E-3.1
Ill APPARATUS
Pipettes Incubator
IV SAMPLING PROCEDURE
Refer to ‘AEROBIC PLATE COUNT' (E-2) SECTION III
V SAMPLE PREPARATIONS
Refer to “ AEROBIC PLATE COUNT” (E-2) SECTION IV
VI PROCEDURE
A. EXAMINATION FOR PRESUMPTIVE COLIFORMS
1. Select appropriate dilutions and for every dilution, transfer 1 ml aliquots into each of 3 LST
tubes.
2. Invert tubes to ensure Durham tubes do not contain gas bubbles.
3. Incubate the tubes at 35°C for 48 hrs.
4. Any tube producing gas is considered positive for the presence of coliforms.
B. CONFIRMATION TESTS FOR COLIFORMS

1. Transfer a loopful of suspension from a positive LST tube into a tube of BGB broth.
2. Invert tubes to ensure Durham tubes do not contain gas bubbles.
3. Incubate the BGB tubes at 35°C for 48 hrs.
4. Examine for gas production.
5. Using the MPN Tables (Appendix A), calculate the MPN of coliforms based on the
proportion of confirmed LST tubes (with gas production) for 3 consecutive dilutions.
C. EXAMINATION FOR PRESUMPTIVE E. COLI

1. Transfer a loopful form each LST tube (with gas production) into a tube of BGB broth,
prewarmed to 44.5°C.
2. Incubate the BGB tubes at 44.5°C for 48 hrs.
3. Examine for gas production at 24 hrs and, if negative, again at 48 hrs.
4. Any tube showing gas production is considered positive for the presence of presumptive
E. coli.
E-3.2
D. CONFIRMATION TESTS FOR E. COLI
1. Subculture all positive BGB tubes by streaking onto plates of EMB agar.
2. Incubate at 35°C for 18-24 hrs.
3. Examine the plates for suspicious E. coli colonies, ie black or dark centred with or
without the greenish metallic sheen.
4. Subculture the suspected E. coli colonies in nutrient broth and incubate at 35°C for
18-24 hrs.
5. Perform the following biochemical tests#:

Indole production
Methyl-Red & Voges-Proskauer tests
Citrate utilization
# R efe r to A p p e n d ix C fo r b io c h e m ic a l te s ts p ro cedu res.
6. Interpret results as follow:-
Indole MR VP Citrate
typical E. coli + + - -
atypical E. coli — + - -
7. Using the MPN Tables (Appendix A), calculate the MPN of E. coli based on the proportion
of BGB tubes in 3 successive dilutions which were shown to contain E. coli.
VII CALCULATION OF MPN

Index 1
Most Probable Number (MPN) = x (450 + W) x
10 W
where W : weight of sample in g

Index : from MPN Tables
VIII BACTERIOLOGICAL LIMITS OF E. COLI FOR FISH/FISHERY PRODUCTS

(COOKED & RAW)
Cooked products : 100 MPN/g
Raw products : 100 MPN/g
REFERENCES
A. Hazzard. (1985). ASEAN Training Course in Fish Quality Control. Training course organised
Chapter: Microbiology in Seafood Quality Control. Section 2:16.
Chapter: Fish quality control microbiology Section 6:88.
See Reference 2 in E-2.
E-3.3
A flow diagram of the examination procedures for coliforms and E. coli is included as the
following figure.
FLOW DIAGRAM OF EXAMINATION PROCEDURES FOR COLIFORMS AND E. COLI
Sample
10–1, 10–2, 10–3 dilutions . . . .
↓
MPN (3 tube method)
Lauryl Sulphate Tryptose Broth (LST)
(1 ml of each dilution into 3 replicate tubes)
35°C/24-48 hrs
gas production
(indicates presumptive coliforms)
Brilliant Green Bile Broth (BGB) Brilliant Green Bile Broth (BGB)
35°C/24-48 hrs 44.5°C ± 0.5°C/24-48 hrs
↓
gas production gas production
↓
confirmation of coliforms presumptive E. coli
Inoculate on Eosin Methylene Blue (EMB)
typical colonies
E—3.4
SALMONELLAE & SHIGELLA
LIM P. Y.
INTRODUCTION
The presence in foods of any serotype of Salmonella is potentially dangerous as a
source of human disease, either directly upon consumption of food, or indirectly through
secondary contamination of utensils, processing equipments or processed foods. A further risk
arises through induction of the carrier state in food-handlers.
I CULTURE MEDIA*
Nutrient broth
Selenite broth
Tetrathionate broth
Desoxycholate citrate agar (DGA)
Xylose lysine deoxycholate (XLD)
Triple sugar iron agar (TSI)
MacConkey agar (MCA)
GN broth
Salmonella anti-sera: Polyvalent “ O” (somatic)
Polyvalent “ H” specific and non-specific (flagellar)
* Refer to Appendix B for methods of media preparation.
II APPARATUS
Pipettes Incubator
Scissors & forceps Agitated water bath
Alcohol (70% v/v) swabs Weighing balance
Plating loops Laminar flow chamber
Inoculating needle Glass slides
Conical flasks or screw-cap jars, 250 ml Petri dish (90 x 15 mm)

Refer to “ AEROBIC PLATE COUNT” (E-2) Section III
IV PROCEDURE
A. RESUSCITATION (PRE-ENRICHMENT)
1. Weigh 50 g of the above sample and put them into a ‘Waring’ blender flask and add
approximately 200 ml of sterile nutrient broth. Homogenise for 1 min at low speed.
Also blend 50 g of above sample with 200 ml of GN broth for Shigella.
2. Incubate at 35°C for 24 hrs; for Shigella incubate at 35°C for 18 hrs.
E-4.1
B. SELECTIVE ENRICHMENT
1. Mix the resuscitated culture gently and add 1 ml each to 10 ml of tetrathionate broth and
10 ml of selenite broth.
2. Incubate the selective enrichment broths at 35°C for 24 hrs.
C. PLATING ON SELECTIVE AGAR MEDIA

1. Each culture of enrichment medium is inoculated onto DCA and XLD agar plates.
Inoculate the same for Shigella from GN broth culture (from Step A-2) on MCA, DCA
and XLD agar plates.
2. Transfer a loopful of culture and streak to obtain isolated colonies.
3. Incubate at 35°C for 24 hrs.
4. Examine the plates for the presence of Salmonella & Shigella colonies.
For Salmonella:
a) On XLD agar: appear as pink colonies with black centres of H2S.
b) On DCA agar: appear as colourless colonies.
For Shigella:
a) On XLD agar: appear as red or pink colour colonies, about 1 mm Ø.
b) On DCA & MCA: appear as opaque or transparent colonies.
D. SCREENING AND BIOCHEMICAL TESTS

1. Pick a suspected colony with inoculating wire and inoculate the TSI agar slant by streaking
the slant and stabbing the butt. Incubate at 35°C for 24 hrs.
2. Salmonella cultures typically produce an alkaline (red) slant and acid (yellow) butt,
with or without production of H2S (blackening of butt) in TSI agar. Shigella
cultures typically produce red slant and yellow butt, with no H2S or gas.
3. Purify TSI cultures by streaking onto MCA and incubate for 24 hrs at 35°C. Typical colonies
appear transparent and colourless, sometimes with a dark centre.
4. Subculture Salmonella colony in nutrient broth and incubate at 35°C for 24 hrs.
Screen typical Shigella cultures in urea agar and motility medium. Shigella is urease
negative and non-motile.
E-4.2
5. Using the nutrient broth culture as inoculum perform the following biochemical tests.
Tests (Salmonella) Results Tests (Shigella) Results

Lysine decarboxylase + Glucose (gas) -
Urease - VP -
Dulcitol + MR +
KCN - Indole + /-
Malonate - Lysine -
Indole - Arginine + /-
VP — Ornithine + /-
MR + Citrate -
Citrate + Mannitol + /-
Lactose - Lactose -
Sucrose -
6. Incubate the tests for 24-28 hrs at 35°C.
7. Note that a large percentage of Salmonella arizonae strains are negative for dulcitol
utilization; positive for malonate and lactose utilization.
8. Perform serological tests for cultures giving reactions typical of Salmonellae & Shigella.
E. SEROLOGICAL CONFIRMATION
1. Emulsify the culture in 2 drops of saline on a clean glass slide.
2. Add one some loopful of polyvalent “ O” antiserum to the first drop only. Use the second
drop as a saline control.
3. Tilt the slide back and forth for 1 minute and examine for agglutination. A positive reaction
is when there is agglutination in the test mixture but not in the saline control.
4. Repeat similarly with polyvalent “ H” antiserum.
5. Salmonella isolates causes agglutination for both antisera.
6. Conduct the serology for Shigella from Step 1 to Step 3.
E-4.3
FLOW DIAGRAM OF EXAMINATION PROCEDURES FOR SALMONELLA
50 g sample + 200 ml nutrient broth

(Pre-enrichment)
35°C/24 hrs
Enrichment in
a) Selenite broth (35°C/24 hrs)
b) Tetrathionate broth (35°C/24 hrs)
Streak onto
a) Desoxycholate citrate agar (DCA)
b) Xylose lysine deoxycholate (XLD)
35°C/24 hrs
Typical colonies
Streak onto TSI
Confirmatory tests:
a) Lysine decarboxylase f) Indole
b) Urease g) VP
c) Dulcitol h) MR
d) Malonate i) Citrate
e) KCN j) Lactose
k) Sucrose
Serology
(Polyvalent “ O” antiserum, “ H” antiserum)
E-4.4
FLOW DIAGRAM OF EXAMINATION PROCEDURES FOR SHIGELLA
50 g sample + 200 ml GN broth

35°C/18 hrs
Streak onto
1) MCA
2) DCA
3) XLD
35°C/24 hrs
↓
TSI
35°C/24 hrs
↓
if alkaline/acid; no H2S, no gas
urease: -
motility: -
V
Confirmatory tests:
Glucose (gas) Arginine
VP Ornithine
MR Citrate
Indole Mannitol
Lysine Lactose
Serology
E–4.5
STAPHYLOCOCCUS AUREUS
LIM P. Y.
INTRODUCTION
Staphylococcus aureus is a common organism on the skin and in the nasal passages of
approximately 50% of the population. Heat treated seafood may become contaminated with
this organism by poor handling, then storage at improper temperatures allows the organism to
multiply and produce its toxin.
This type of food poisoning may be avoided by practising strict personal hygiene,
thorough cleaning and disinfection of equipment, and storage of susceptible food at
temperatures below 10°C or above 60°C.
Examination of a product for S. aureus does not guarantee protection against

staphylococcal food poisoning because the organism may be killed, without destruction of the
heat stable enterotoxin produced during growth of the organism. A direct microscopic smear of
the food may be helpful, as direct detection of toxin in food requires methods which are too
involved for routine use. A smear reveals viable and killed cells of staphylococci.
I CULTURE MEDIA*
Baird Parker medium
Brain heart infusion broth (BHI)
Citrated human plasma
Trypticase soy broth + 10% NaCl (TSB)
II APPARATUS
‘Waring’ blender & flasks Autoclave Test-tubes
Pipettes Incubator Plating loops

Refer to “ AEROBIC PLATE COUNT” (E-2) Section IV
E-5.1
V PROCEDURE
1. Select appropriate dilutions and for every dilution, transfer 1 ml aliquots into each of 3 TSB
tubes.
3. The presence of turbidity indicates presumptive S. aureus.
4. Streak a loopful of the culture from a positive tube onto Baird Parker agar plate.
5. Incubate the plates at 35°C for 48 hrs.
6. Typical colonies of S. aureus on Baird Parker agar appear as smooth, black, convex and
shiny with narrow white entire margins and are surrounded by clear zones extending into
the opaque medium.
7. Subculture all suspected colonies in BHI broth and incubate at 35°C for 24 hrs.
8. Transfer 0.5 ml of the broth culture into a test-tube and add 1 ml of citratedhuman plasma.
Mix by gentle rotation of the tube.
9. Incubate at 35°C for about 6 hrs, and if negative, examine again after 24 hrs.
10. A 3+ or 4+ clot formation is considered a positive reaction for S. aureus. A 3 +

reaction refers to formation of a large organized clot and a 4+ reaction is when the entire
contents of the tube coagulate and is not displaced when the tube is inverted. (See
illustration overleaf).
11. Using the MPN Tables (Appendix A), calculate the MPN of S. aureus based on the
proportion of confirmed turbid TSB tubes for 3 consecutive dilutions.
VI CALCULATION OF MPN
Index 1
10 W
where W : weight of sample in g.

Index : from MPN tables.
E-5.2
TYPES OF COAGULASE TEST REACTIONS
NEGATIVE NO EVIDENCE OF FIBRIN FORMATION

1 + POSITIVE SMALL UNORGANIZED CLOTS
2 + POSITIVE SMALL ORGANIZED CLOT
3 + POSITIVE LARGE ORGANIZED CLOT
4 + POSITIVE ENTIRE CONTENT OF TUBE COAGULATES AND IS NOT DISPLACED
WHEN TUBE IS INVERTED
VII BACTERIOLOGICAL LIMITS OF S. AUREUS FOR FISH/FISH PRODUCTS

(COOKED & RAW)
Cooked products : 100 MPN/g
Raw products : 250 MPN/g
REFERENCES
by HAWKAID, Hawkesbury Agricultural College Research Development Co. Ltd. Chapter:
Microbiology in seafood quality control. Section: 68, 114 & 115.
E-5.3
A flow diagram of the examination procedures for Staphylococcus aureus
is included as the following figure.
FLOW DIAGRAM OF EXAMINATION PROCEDURES FOR STAPHYLOCOCCUS AUREUS
Sample
10–1, 10– 2, 10– 3 dilutions
MPN (3 tube method)

Trypticase Soy Broth + 10% NaCI
35°C/48 hrs
positive tubes showing turbidity

streak onto Baird Parker medium
35°C/48 hrs
typical colonies
Brain Heart Infusion broth (BHI)

35°C/24 hrs
Coagulase Test
0.5 ml of broth culture
added to 1 ml of citrated plasma
35°C/6 hrs
3 + / 4 + clot formation
FAECAL STREPTOCOCCI
LIM P.Y.
INTRODUCTION
Streptococci are gram positive cocci, sometimes coccobacilli, arranged in chains. This
group of streptococci resides in the intestine of warm-blooded animals. They are bile resistant
and capable of growth at 45°C.
Faecal streptococci form part of the microflora of many food without necessarily indicating
poor hygiene. They are found in many fermented food, such as cheese and raw sausage, and
often take part in the fermentation process. However, in meat products which have received a
severe heat process, the presence of excess numbers of faecal streptococci indicates
unhygienic handling and/or faulty storage.
I CULTURE MEDIA*
Azide dextrose broth (ADB)
Bromocresol purple azide broth
* R e f e r t o A p p e n d i x B f o r m e t h o d s o f m e d i a p r e p a r a t i o n .
II APPARATUS
Pipettes Incubator

Refer to ‘AEROBIC PLATE COUNT” (E-2) SECTION III
Refer to “ AEROBIC PLATE COUNT” (E-2) SECTION IV
V PROCEDURE
1. Select appropriate dilutions and for every dilution, transfer 1 ml aliquots into each of 3 ADB
tubes.
3. The presence of turbidity indicates presumptive faecal streptococci.
E -6 .1
4. Transfer a loopful of suspension from a positive ADB tube into a tube of bromocresol
purple azide broth.
6. The bromocresol purple azide broth turning purple red confirms the presence of faecal
streptococci.
7. Using the MPN tables (Appendix A), calculate the MPN of faecal streptococci based on the
proportion of confirmed positive bromocresol purple azide broth tubes for 3 consecutive
dilutions.
VI CALCULATION OF MPN
Index 1
10 W
where W : weight of sample in g.

Index : from MPN tables.
VII BACTERIOLOGICAL LIMITS OF FAECAL STREPTOCOCCI FOR FISH/FISHERY

PRODUCTS (COOKED & RAW)
Cooked products : —
Raw products : 1,000 MPN/g
REFERENCES
A. Hazard. 1985. ASEAN Training Course in Fish Quality Control. Training course organised by
HAWKAID, Hawkesbury Agricultural College Research and Development Co. Ltd.
Chapter: Microbiology in seafood quality control. Section 2: 17 & 28.
Chapter: Fish quality control microbiology. Section 6. Page 88.
E-6.2
A flow diagram of the examination procedures for Faecal Streptococci is included as the
following figure.
FLOW DIAGRAM OF EXAMINATION PROCEDURES OF FAECAL STREPTOCOCCI
Sample
10– 1 , 10– 2, 10– 3 dilutions
MPN (3 tube method)

Azide Dextrose Broth
35°C/24 hrs
Positive tubes showing turbidity

inoculate into
Bromocresol Purple Azide Broth
35°C/24 hrs
↓
Broth turns purple red
Faecal streptococci confirmed
E-6.3
VIBRIO CHOLERA
LIM P. Y.
INTRODUCTION
Cholera is an acute specific infection caused by the organism, Vibrio cholera. Diagnosis
may be confirmed by the presence of large numbers of the comma-shaped bacilli on direct
microscopic examination of a faecal or vomitus smear, and by the isolation of the organism on
culture.
Fish and shellfish have been identified as vehicles of cholera. Large numbers of V. cholera
must usually be ingested to cause cholera, thus problems often occur when poor handling and
inadequate refrigeration have allowed the organism to multiply.
I CULTURE MEDIA*
Alkaline peptone water (pH 8.6-9.0) Phenylalanine agar (PPA)
Andrade peptone water SIM medium
Aesculin broth Simmons citrate agar
Decarboxylase medium base Thiosulphate citrate bile
Koser citrate medium salts sucrose agar (TCBS)
MRVP medium Triple sugar iron agar (TSI)
Nutrient agar (+ 3% NaCI) Sodium chloride (NaCI)
Nutrient gelatin
a) 1% solution (w/v) of each of the following amino acids:-

L-arginine HCl
L-lysine HCI
L-ornithine HCl
b) 1% solution (w/v) of each of the following sugars:-

Arabinose Lactose Melibiose
Glucose Mannitol Salicin
Inositol Mannose Sucrose
II CHEMICAL REAGENTS#
a) Tetramethyl-p-phenylenediamine di-HCl aq. soln. (1% w/v)
b) Kovac’s reagent
c) 0.1N HCI
d) Methyl red solution
e) KOH solution (40% w/v)
f) α-naphthol solution (5% w/v)
g) FeCI3 aq. soln. (10% w/v)
# Refer to A ppendix D fo r m ethods o f reagent preparation.
E-7.1
Ill APPARATUS
Pipettes Incubator
Plating loops
V PROCEDURE
1. Weigh about 50 g of the sample and add approximately 200 ml of alkaline peptone water in
a ‘Waring’ blender flask. Blend for 1 min at low speed.
2. Incubate at 35°C for 6-8 hrs.
3. At the end of the incubation period, transfer a loopful obtained from the pellicle (surface
growth) onto TCBS agar and streak to obtain isolated colonies.
4. Incubate the plates at 35°C for 18-24 hrs.
5. V. cholera colonies on TCBS agar appear as large, smooth and yellow.
6. Screen isolates with the following tests*:-
Tests Results
TSI acid slant/acid butt; no gas; no H2S
Indole (SIM) +
Motility (SIM) +
Lysine decarboxylase +
Peptone water (+3% NaCI) growth
* Refer to Appendix C for biochemical tests procedures.
7. From the TSI slant, inoculate a nutrient agar (+3% NaCI) slant and incubate at 35°C for 24
hrs.
E-7.2
8. Perform the oxidase test from the nutrient agar slant and use the peptone water culture as
inoculum for the following biochemical tests*.
Tests Results
Oxidase +
Lysine +
Ornithine +
Arginine —
Sucrose +
Mannitol +
Inositol -
MR + w (Reaction delayed & weak)

VP + / - (Indefinite)
PW + 0% NaCI +
PW + 3% NaCI +
PW + 7% NaCI d (16-84% strains positive)
PW + 9% NaCI —
PW + 11% NaCI —
9. Carry out the following confirmatory biochemical tests*:-
Tests Results
Citrate + w (Reaction delayed & weak)
Phenylalanine —
Gelatin (5°C) +
Gas from glucose —
Lactose —
Arabinose —
Mannose +
Salicin —
Melibiose —
Aesculin -
E-7.3
10. Serological agglutination tests are performed on confirmed isolates using polyvalent O
anti-serum and Ogawa and Inaba anti-sera.
VI BACTERIOLOGICAL LIMITS OF VIBRIO CHOLERA FOR FISH/FISHERY PRODUCTS

(COOKED & RAW)
This organism should not be detected in 50 g sample.
REFERENCES
Chapter: Microbiology In Seafood Quality Control. Section 6: 68 & 77.
A flow diagram of the examination procedures for V. cholera is included

as the following figure.
FLOW DIAGRAM OF EXAMINATION PROCEDURES FOR VIBRIO CHOLERA
50 g sample + 200 ml Alk.peptone water (pH 8.6-9.0)
(enrichment stage)
35°C/6 hrs
streak onto TCBS

35°C/24 hrs
yellow colony on TCBS
i) TSI slant : A/A (no gas, no H2S)

ii) SIM : indole + motility +
iii) L-lysine HCI : +
iv) Peptone water + 3% NaCI : growth
(use as inoculum)
Na (+3% NaCI) slant — for oxidase test
Oxidase +
L-lysine HCI +
L-ornithine HCI +
E-7.4
L-arginine HCI —
Sucrose +
Mannitol +
Inositol -
MR +w (Reaction delayed & weak)
VP + /- (Indefinite)
PW + 0% NaCI +
PW + 3% NaCI +
PW + 7% NaCI d (16-84% strains positive)
PW + 9% NaCI -
PW + 11% NaCI —
Y
Confirmatory biochemical tests
Citrate +w (Reaction delayed & weak)

Phenylalanine -
Gelatin (5°C) +
Gas from glucose -
Lactose -
Arabinose -
Mannose +
Salicin -
Aesculin -
Melibiose -
serology for V. cholera
E–7.5
VIBRIO PARAHAEMOLYTICUS
LIM P. Y.
INTRODUCTION
Food poisoning due to V. parahaemolyticus is a food-borne infection resulting from
the ingestion of a large number of this organism (about 106-109 viable cells). The major
symptoms are diarrhoea and abdominal pain with headache, fever and vomiting also occurring.
The organisms are excreted during the acute stage of the illness after which they decrease
rapidly.
The differentiation of V. parahaemolyticus from other pathogenic species of Vibrio is

based mainly on salt tolerance, Voges-Proskauer reaction, fermentation of sucrose and growth
at 43°C.
I CULTURE MEDIA*
Glucose salt teepol broth (GSTB)
Modified Wagatsuma agar
Thiosulphate citrate bile salts sucrose agar (TCBS)
MRVP medium
Andrade peptone water
Phenylalanine agar (PPA)
Bacto-peptone (PW)
Decarboxylase medium base
Nutrient gelatin
Aesculin broth
SIM medium
Nutrient agar (+3% NaCI)
Sodium chloride (NaCI)
* R efe r to A p p e n d ix B fo r m e th o d s o f m e d ia p re p aration .
a) 1% solution (w/v) of each of the following amino acids:-

L-arginine HCI
L-lysine HCI
L-ornithine HCI
b) 1% solution (w/v) of the following sugars:-

Arabinose Mannitol Salicin
Glucose Mannose Sucrose
Lactose Melibiose
E-8.1
II CHEMICAL REAGENTS**
Tetramethyl-p-phenylenediamine di-HCI aq. soln. (1% w/v)
a) Kovac’s reagent d) 01.N HCI
b) Methyl red solution e) KOH solution (40% w/v)
c) α-naphthol solution (5% w/v) f) FeCI3 aq. soln. (10% w/v)
** R efe r to A p p e n d ix D fo r m e th o d s o f re a g e n t p re p a ra tio n .
Ill APPARATUS
Pipettes Incubator
Plating loops
Refer to “ AEROBIC PLATE COUNT” (E-2) Section III.
V SAMPLE PREPARATION
Refer to “ AEROBIC PLATE COUNT” (E-2) Section IV.
VI PROCEDURE
1. Select appropriate dilutions and for first dilution, transfer 10 ml aliquots into each of 3
tubes of double strength GSTB.
2. For each of the next 2 further dilutions, transfer 1 ml aliquots into each of 3 tubes of single
strength GSTB.
3. Incubate the tubes at 35°C for not more than 18 hrs.
4. Transfer a loopful of suspension from the top 1 cm of a positive GSTB tube onto a TCBS
plate and streak to obtain isolated colonies.
5. Incubate the plates at 35°C for 18 hrs.
6. Examine the plates for typical V. parahaemolyticus colonies which are large and
blue-green with a dark centre.
7. Screen suspected isolates by inoculating the following media* and incubate at 35°C for 24
hrs.
TSI agar K/Acid (no gas; no H2S)

Indole (SIM) +
Motility (SIM) +
L-lysine HCI +
* R efe r to A p p e n d ix C fo r b io c h e m ic a l tes ts p ro c e d u re s .
E-8.2
8. Inoculate the TSI culture into peptone water (+3% NaCI) and nutrient agar (+3% NaCl)
slant and incubate at 35°C for 24 hrs.
9. Perform the oxidase test from the nutrient agar slant and use the peptone water culture as
inoculum for the following biochemical tests.*
Oxidase +
Voges-Proskauer -
Sucrose -
Mannitol +
Peptone water (PW) + 0% NaCI -
Peptone water (PW) + 3% NaCI +

Peptone water (PW) + 11% NaCI -
* R efer to A p p e n d ix C fo r b io c h e m ic a l te s ts p ro ced u res.
10. Carry out the following confirmatory biochemical tests*
Methyl Red (MR) +

Citrate +
L-arginine HCl -
L-ornithine HCI +
Phenylalanine (PPA)
Nutrient gelatin (5°C) ■
Gas from glucose -
Lactose -
Arabinose +
Mannose +
Mannitol +
Salicin -
Aesculin +
Melibiose —
* R efer to A p p e n d ix C fo r b io c h e m ic a l te s ts p ro ced u res.
11. Calculate the MPN of V. parahemolyticus based on the proportion of positive GSTB tubes
which are. confirmed for the presence of parahaemolyticus. (See below)
E-8.3
VII CALCULATION OF MPN
Index 1
10 W
where W : weight of sample in g

Index : from MPN Tables (Appendix A)
REFERENCES
A. Hazzard. (1985). ASEAN Training Course in Fish Quality Control. Training course
organised by HAWKAID, Hawkesbury Agricultural College Research and Development
Co. Ltd. Chapter: microbiology in seafood quality control. Section 6: P. 69-70.
Isolation and identification of Vibrio parahaemolyticus. Bacteriological analytical manual.

Jan. 1969.
A flow diagram of the examination procedures for V. parahaemolyticus is included as

the following figure.
FLOW DIAGRAM OF EXAMINATION PROCEDURES FOR V. PARAHAEMOLYTICUS
Sample
i
10– 1 , 10– 2, 10– 3 dilutions . . . .
Y
MPN (3 tube method)
Glucose Salt Teepol Broth (GSTB)

i) double strength — 3 x 10 ml of 10–1
ii) single strength — 3 x 1ml of 10–2
iii) single strength — 3 x 1ml of 10–3
35°C/< 18 hrs
Y
streak onto
Thiosulphate Citrate Bile Salts Sucrose (TCBS)
35°C/18 hrs
Y
blue-green colony on TCBS agar
Y
i) TSI slant K/A (no gas, no H2S)
ii) SIM medium Indole: + Motility: +
iii) L-lysine HCI +
E-8.4
↓
Inoculate i) Peptone water (+3% NaCI) (as inoculum)

ii) Nutrient agar (+3% NaCI) slant (for oxidase test)
i) Oxidase +
ii) Voges-Proskauer -
iii) Sucrose -
iv) Mannitol +
v) PW + 0%NaCI
PW + 3%NaCI +
PW + 7%NaCI +
PW + 9%NaCI +
PW + 11%NaCI
↓
confirmatory biochemical tests
Methyl Red (MR) +
Citrate +
L-arginine HCI -
L-ornithine HCI +
Phenylalanine (PPA) -
Nutrient gelatin (5°C) +
Melibiose -
Gas from glucose -
Lactose -
Arabinose +
Mannose +
Mannitol +
Salicin -
Aesculin +
↓
Kanagawa reaction test (if required) (see next page)
E-8.5
KANAGAWA REACTION OF V. PARAHAEMOLYTICUS
APPLICATION
The Kanagawa reaction tests for the presence of specific haemolysis on Wagatsuma agar.
A positive reaction has been found to correlate closely with the pathogenicity of
V. parahaemolyticus isolates. The isolates that have caused illness in humans are almost
always Kanagawa-positive, although isolates from seafood are almost always Kanagawa-
negative.
PROCEDURE
1. Subculture the isolate into 3% NaCI peptone water and incubate at 35°C for 18 hrs.
2. Spot a loopful of this culture onto a freshly prepared, dried modified Wagatsuma agar
plate. Several spottings may be made on the same plate.
3. Incubate at 35°C for 18 ± 2 hrs.
4. A positive test consists of β-haemolysis: a zone of transparent clearing of the blood cells
around the colony.
5. It is very important to remember that only observations within 24 hrs is valid in this test.
E-8.6
APPENDIX
APPENDIX A
MOST PROBABLE NUMBERS (MPN) PER 1 G OF SAMPLE, USING 3 TUBES WITH
EACH OF 0.1, 0.01 AND 0.001 G PORTIONS
MPN INDEX TABLE

No. of Tubes Giving Positive Reaction out of MPN Index per
3 of 1 ml 3 of 0.1 ml 3 of 0.01 ml 10 ml
Each Each Each
0 0 0 <3
0 0 1 3
0 0 2 6
0 0 3 9
0 1 0 3
0 1 1 6.1
0 1 2 9.2
0 1 3 12
0 2 0 6.2
0 2 1 9.3
0 2 2 12
0 2 3 16
0 3 0 9.4
0 3 1 13
0 3 2 16
0 3 3 19
1 0 0 4
1 0 1 7
1 0 2 11
1 0 3 15
1 1 0 7
1 1 1 11
1 1 2 15
1 1 3 19
1 2 0 11
1 2 1 15
1 2 2 20
1 2 3 24
1 3 0 16
1 3 1 20
1 3 2 24
1 3 3 29
2 0 0 9
2 0 1 14
2 0 2 20
2 0 3 26
2 1 0 15
2 1 1 20
2 1 2 27
2 1 3 34
2 2 0 21
2 2 1 28
2 2 2 35
2 2 3 42
2 3 0 29
2 3 1 36
2 3 2 44
2 3 3 53
3 0 0 23
3 0 1 39
3 0 2 64
3 0 3 95
3 1 0 43
3 1 1 75
3 1 2 120
3 1 3 160
3 2 0 93
3 2 1 150
3 2 2 210
3 2 3 290
3 3 0 240
3 3 1 460
3 3 2 1,100
3 3 3 >2,400
APPENDIX B
M E D IA PR E P A R A TIO N M E T H O D S
B1 BUTTERFIELD’S BUFFERED PHOSPHATE DILUENT
1. Stock solution
Dissolve 6.8 g KH2PO4 in 100 ml H20, adjust pH 7.2 with ca 35 ml -1N NaOH and
dilute to 200 ml. Store in refrigerator.
2. Diluent
Dilute 1.25 ml stock solution to 1 litre with distilled H20. Prepare diluent with this
solution, dispensing enough to allow for losses during autoclaving. Autoclave for
15 mins at 121°C.
B2 MEDIA PREPARATION
For the method of preparing the following media, refer to the respective manufacturer’s
manual. Available manuals: BBL, Difco, Merck & Oxoid.
Aesculin broth
Andrade peptone water
Azide dextrose broth (ADB)
Bacto-peptone (PW)
Baird Parker medium
Brain heart infusion broth (BHI)
Brilliant green bile (2%) broth (BGB)
Bromocresol purple azide broth
Decarboxylase medium base
Desoxycholate citrate agar (DCA)
Eosin methylene blue agar (EMB)
GN broth
Lauryl sulphate tryptose broth (LSB)
MacConkey agar (MCA)
Modified Wagatsuma agar
MRVP medium
Nutrient agar
Nutrient broth
Nutrient gelatin
Phenylalanine agar (PPA)
Plate count agar (PCA)
Selenite broth
SIM medium
Tetrathionate broth
Thiosulphate citrate bile salts sucrose agar (TCBS)
Trypticase soy broth (TSB)
Xylose lysine deoxycholate (XLD)
APPENDIX C
B IO C H E M IC A L TE S TS IN D IA G N O S T IC M IC R O B IO L O G Y
C1 AESCULIN HYDROLYSIS
Inoculate Aesculin broth and examine daily up to 7 days for blackening, this indicates
hydrolysis of the aesculin. Alternatively, inoculate Aesculin agar and look for blackening
in and around the bacterial growth.
C2 CARBOHYDRATE BREAKDOWN
Inoculate the Andrade peptone water sugar and examine after 24 hrs of incubation. Acid
production is indicated by a change in the colour from colourless to pinkish or reddish.
Formation of gas is indicated by a bubble in the inverted Durham tube.
C3 CITRATE UTILIZATION
Method 1.
Inoculate Koser citrate broth and incubate at 35°C for at least 24 hrs. Examine for
turbidity.
Turbidity — citrate utilized

No turbidity — citrate not utilized
Method 2.
Inoculate by making a single streak over the surface of a slope of Simmons citrate.
Examine for growth and colour change.
Blue colour and streak of growth — citrate utilized

Original green colour — citrate not utilized
C4 COAGULASE TEST
Method 1. Slide test

Emulsify a colony from a culture plate in a drop of saline on a microscope slide.
Emulsify the culture well to make a milky suspension of the organisms. Mix a loopful of
human plasma into the drop of bacterial suspension.
Tilt the slide back and forth and observe for formation of granular precipitate of white
clumps. Clumping of bacteria indicates presence of coagulase (coagulase-positive),
and usually occurs within 15 to 20 seconds. The test is considered negative if clumping
is not observed within 2 to 3 minutes.
Method 2. Tube test
To 0.5 ml of a 24 hrs broth culture of the organism in a test-tube, add 1 ml of human
plasma. Mix by gentle rotation of the tube, avoiding stirring or shaking of the mixture.
Incubate at 35°C for 1 to 4 hrs.
Observe for formation of a visible clot.
C5 DECARBOXYLASE REACTIONS
Inoculate tubes of the Decarboxylase medium containing 1% (w/v) solution of amino
acid (L-arginine HCl or L-lysine HCI or L-ornithine HCI) and incubate at 35°C. Examine
daily for up to 4 days. Decarboxylation is indicated by a purple colour, whereas the
control and negative tubes are yellow.
C6 GELATIN HYDROLYSIS
Inoculate Nutrient gelatin and incubate at 35°C for up to 14 days. For every 2 to 3 days,
cool in a refrigerator for half an hour and then examine for liquefaction. Set up a control
tube of uninoculated medium in parallel.
C7 HYDROGEN SULPHIDE PRODUCTION
Method 1.
Inoculate a tube of Triple sugar iron (TSI) agar by stabbing the butt and streaking the
slope. Observe for blackening due to H2S production.
Method 2.
Inoculate a tube of SIM medium by stabbing into the butt. Observe for blackening due to
H2S production.
C8 INDOLE PRODUCTION
Inoculate a tube of SIM medium by stabbing into the butt. Incubate for 48 hrs at 35°C.
Add Kovac’s reagent down the side of the tube. A red colour in the reagent layer
indicates indole.
C9 MOTILITY
Method 1.
Transfer a loopful of a young broth culture of the organism to a clean microscope slide.
Cover with a cover-slip. Examine for motility using a high-power dry objective and
reduced illumination.
Method 2.
Inoculate a tube of SIM medium by stabbing to a depth of 1 cm from the bottom. After
incubation at 35°C for 24 hrs, examine the growth pattern of the organism.
A motile organism migrates from the stab line and diffuses into the medium, causing a
turbidity; or it may exhibit fuzzy streaks of growth. Growth of a non-motile organism is
concentrated along the stab line, with the surrounding medium remaining clear.
C10 METHYL-RED REACTION
Inoculate MRVP broth and incubate at 35°C for 24 to 48 hrs. Add 2 drops of methyl red
solution, shake and examine.
red colour — +
orange — ±
yellow —
C11 OXIDASE ACTIVITY

On a piece of filter paper in a petri-dish, place 2 to 3 drops of the oxidase reagent (1 %
w/v tetramethyl-p-phenylenediamine di-HCI aq. solution); do not allow the drops to dry
on the plate. The test organism is removed with a platinum wire and smeared across the
surface of the impregnated paper. A positive reaction is shown by the development of a
dark purple colour within 10 seconds.
C12 PHENYLALANINE DEAMINATION
Method 1.
Inoculate Malonate-phenylalanine medium and incubate for 24 hrs a t 35°C. Acidify with
0.1-0.2 ml of 0.1N HCI; add 0.2 ml 10% FeCI3 aq. solution; shake and observe
immediately any colour change. A positive reaction is indicated by a green colour which
quickly fades.
Method 2.
Inoculate heavily a Phenylalanine agar slope. Incubate overnight and run 0.2 ml 10%
FeCI3 aq. solution over the growth. A positive result gives a green colour on the slope
and in the free liquid at the base.
C13 VOGES-PROSKAUER (VP) REACTION

Inoculate MRVP broth and incubate at 35°C for 24 hrs. After completion of the methyl
red test, add 0.6 ml of 5% α -naphthol solution followed by 0.2 ml 40% KOH solution.
Shake, slope the tube (to increase the size of the air/liquid interface) and examine after
15 minutes and 1 hr. A positive reaction is indicated by a strong red colour.
C14 TRIPLE SUGAR IRON (TSI) TEST

With the inoculating needle, touch a well-isolated colony on a culture plate and stab the
needle into the deep of the tube to a depth of 1 cm from the bottom. Remove the needle
from the deep and streak the slant surface. Incubate at 35°C for 24 hrs.
Record the TSI results with the slant reaction first followed by the deep reaction,
separated by a slash mark (slant reaction/deep reaction). The slant reaction involves the
presence or absence of acidity (carbohydrate fermentation). When interpreting the deep
reaction observe for:-
a) absence or presence of acidity

b) presence of CO2 and H2 gases, as evident by a splitting of the medium, a single
gas bubble, complete displacement of the medium from the bottom of the tube
leaving a clear area, or a slight indentation of the medium from the side of the tube.
c) presence of a black precipitate indicating that H2S gas was produced.
Use the following standard abbreviations to record the TSI results:-
Acidity (yellow) — A
Alkalinity (purplish/red) — K
CO2 and H2 gases — gas

Hydrogen sulphide — H2S
No change — NC
APPENDIX D
P R E P A R A TIO N M E T H O D S FOR R EA G EN TS
D1 KOVAC’S (1928) REAGENT FOR INDOLE

p-dimethylaminobenzaldehyde 5g
Iso-Amyl alcohol 75 ml
Conc. HCI 25 ml
Dissolve the aldehyde in the alcohol by gently warming in a water bath (about 50-55°C).
Cool and add the acid. Protect from light and store at 4°C.
Note: The reagent should be light yellow to light brown in colour; some samples of amyl alcohol are
unsatisfactory, and give a dark colour with the aldehyde.
D2 METHYL RED SOLUTION

Methyl red 0.04 g
Ethanol 40 ml
Distilled water to 100 ml
Dissolve the methyl red in the ethanol and dilute to volume with distilled water.
D3 α -NAPHTHOL SOLUTION
5%(w/v) α -naphthol in ethanol
The solution should not be darker than straw colour; if necessary the α -naphthol should
be redistilled (Fulton, Halkias & Yarashus, 1960).
D4 OXIDASE TEST REAGENT

1%(w/v) tetramethyl-p-phenylenediamine dihydrochloride aq. solution
The reagent should be colourless and be stored in a glass-stoppered bottle, protected

from light, at 4°C. The solution should not be used if it becomes deep blue. The
autoxidation of the reagent may be retarded by the addition of 1% ascorbic acid (Steel,
1962b). If ascorbic acid is not used to delay the process of autoxidation, the solution
should be freshly prepared each week.
REFERENCE
COWAN, S.T. (1974) ‘Manual for the identification of medical bacteria’ 2nd Ed Cambridge
University Press.

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LABORATORY MANUAL ON ANALYTICAL METHODS AND

PROCEDURES FOR FISH AND FISH PRODUCTS

MARINE FISHERIES RESEARCH DEPARTMENT

Southeast Asian Fisheries Development Center

Secretariat: Liaison Office

Copyright © 1987. Marine Fisheries Research Department

PART A. DETERMINATION OF PHYSICAL PROPERTIES OF MEAT

A-2 Determination of ash

A -4 Measurement of free and expressible drips

A -5 Fish protein extractibility and its determination

A -6 Viscosity of fish meat sol

PART B. DETERMINATION OF CHEMICAL PROPERTIES OF MEAT

B-1 Protein determination by Kjeldahl Method

B-2 Protein determination by Biuret Method (Modified by Umemoto)

B-3 Determination of trimethylamine oxide (TMAO-N), trimethylamine (TMA-N),

B-4 Determination of DMA-N by Dyer’s Colorimetric Method using copper

B-5 Determination of formaldehyde in fish meat using Nash’s Reagent

B-7 Freshness testing paper

C-1 Significance of analysis of lipids

C-2 Extraction of lipids (modified Folch’s Method)

C-3 Determination of total lipid content

C -4 Determination of phospholipid content

C -5 Determination of acid value

C-6 Determination of saponification value

C -7 Determination of peroxide value

C -8 Determination of thiobarbituric acid (TBA) number

C -9 Determination of methyl esters of fatty acids by gas chromatographic

C-10 Determination of the degree of lipid oxidation by chromatography

C-11 Preparation of methyl esters by Boron Triflouride Method

PART D. ANALYSIS OF ADDITIVES

D-1 Detection of polyphosphates

D-2 Determination of monosodium L-glutamate (MSG) content in fish jelly

D-3 Determination of sugar (sucrose) by Somogyi’s Method

D-4 Determination of starch

D-5 Determination of salt

E-1 Handling of food samples

E-2 Aerobic plate count

E-3 Coliforms and Escherichia coli

E-4 Salmonellae and Shigella

E-5 Staphylococcus aureus

E-6 Faecal streptococci

E-7 Vibrio cholera

E-8 Vibrio parahaemolyticus

B Media preparation methods

C Biochemical tests in diagnostic microbiology

D Preparation methods for reagents

a) the form in which water is present

Method 2: infra-red balance (Kett, model F-1A).

Method 3: microwave moisture checker (Anritsu, model K377C).

III ANALYTICAL PROCEDURE AND CALCULATION

where W 1 = weight (g) of sample before drying.

METHOD 2: INFRA-RED METHOD

(b) Calculate similarly the oven method i.e.;

where W 1 = weight of sample before drying.

METHOD 3: MICROWAVE METHOD

6. Press the start switch to activate the drying.

8. Press the readout button to obtain the dried weight.

9. Repeat additional 30 sec at 300w until dried weight is constant.

This method is applicable to all food materials.