3.3.8 bath for 2hours. The mixture was

Determination of Polycyclic Aromatic Hydrocarbon (PAH) (AOAC, 2000)

A known weight of the fish sample was taken into 500ml
flask. 200ml ethanol, 35ml 50% KOH and ca2g Na2S.9H2O was
simultaneously added to the sample and refluxed in water bath for 2hours. The
mixture was cooled and mixed with 150ml n-hexane with occasional swirling. The
mixture was transferred into 500ml separating funnel containing 150ml distilled
water. The mixture was vigorously shaken and allowed to separate into layers.
The organic layer was separated while the aqueous layer was extracted twice and
combined with the organic layer. The combined organic layer was washed with
100ml water and dried over anhydrous sodium sulphate. The dried solution was
evaporated to 3-5ml. 20µL of the resultant solution was injected into HPLC and
was run for 25 minutes. Individual component were identified by retention time
by comparing with respective standards using Class VP Shimadzu software.
Concentration of each component was obtained from the software-generated data
by external standard method. Concentration was calculated as:

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µg PAH/litre= A x C x 2

x D x 1000

Where A= ng pesticide in sample injected, B= extract
volume, 20µL, C= volume of sample, (20µL), and D= volume of water taken for
analysis (500ml).

3.3.9 Quality and Nutritional Analysis of Retort Pouch

The smoked and steamed milkfish steaks in oil and dry were
subjected to quality evaluation (TBA, FFA, TMA, TVN, pH, texture and instrumental
colour) and nutritional profiling (proximate composition, minerals, fatty acid
methyl ester (FAME) and amino acid), after the thermal processing. These
samples were stored at ambient temperature (28oC) to determine the
keeping quality. Samples were periodically analysed once every month to
determine changes in pH, texture, colour, thiobabituric acid (TBA), free fatty
acid (FFA), trimethyl amine (TMA), total volatile nitrogen (TVN) as well as the
proximate composition. Analyses were done in triplicates for all the samples. Determination of moisture (AOAC, 2000)

10g of the homogenised sample was weighed in a
pre-dried and weighed petri dish on an electronic balance. The samples were
oven dried at 80oC for 15 hours and cooled in a desiccator after
which they were oven dried again at 100oC, cooled and weighed
periodically until constant weight was obtained. The moisture content was
calculated and expressed in percentage.

%Moisture= Loss in weight x 100                                                                                                                                  

                        Weight of sample Protein Estimation (AOAC, 2000)

A known quality of the homogenised wet sample was transferred
into a kjedahl flask of 100ml capacity. Few glass beads and a pinch of
digestion mixture and 10ml of concentrated sulphuric acid were added. This was
digested over a burner until solution turned colourless. The digested mixture
was cooled after which distilled water was added in small quantities with
intermittent shaking and cooling until the addition of water no longer
generated heat. The solution was transferred quantitatively into a 100ml
standard flask and made up to the volume with distilled water. 5ml of the made
up solution was pipetted and transferred to the reaction chamber of the
Micro-Kjedahl distillation apparatus. 10ml 40% sodium hydroxide was added along
with 2 drops of phenolphthalein indicator. These were washed down with little
volume of distilled water. 10ml of 2% boric acid containing a drop of Tashiro’s
indicator in a conical flask was placed at the tip of the distillation
apparatus in such a way that the tip touched the solution inside the conical
flask. Distillation was done for 4 minutes and ammonia liberated was absorbed
into the boric acid. This was observed as the solution turned green from the
initial pink colour. The amount of ammonia liberated was determined by
titration with 0.01N standardised sulphuric acid until there was a colour
change from green back to the initial pink colour. Total protein was calculated

% Protein= X x 0.14 x V x 6.25 x 100

x W x 1000


V= Total Volume of Digest (100ml)

V1= Volume of Digest for Distillation

W= Weight of sample for Digestion

X= Titre Value
Estimation of Crude fat (AOAC, 2000)

1g of the moisture free sample was taken into a
thimble plugged with cotton and placed inside a flat bottom flask. Extraction
was done with petroleum ether at 90oC in a Soxhlet apparatus for
about 2hours 30minutes after which excess solvent was evaporated at 150oC.
The fat was dried at 100oC to constant weight. The crude fat was
calculated and expressed as percentage.

% Crude fat = weight of fat x 100

of the sample
Determination of ash content (AOAC, 2000)

2g of the moisture free sample was transferred into a
pre-heated and weighed silica crucible. The samples were then charred at low
flame on a clay triangle. The charred material was kept inside a muffle furnace
set at 600oC for 6-8 hours until a white ash was obtained. Crucibles
were weighed after cooling in a desiccator and percentage of ash was

% Ash= weight of residue x 100

of the sample Determination of Mineral (AOAC)

Finely powdered dried fish sample was ashed in a
muffle furnace at 600oC. The sample was cooled in a desiccator. The
acquired ash was dissolved in 2ml concentrated HCl and transferred into a 50ml
standard flask. Dissolution was repeated three times with concentrated HCl. The
crucible was washed with distilled water and transferred to the flask. Volume
was made up with distilled water. The made up solution was filtered into sample
bottle with Whatman No 1 filter paper. The standard samples including reagent blank
were sprayed into a flame photometry. The standard solutions for the different
minerals were sprayed over the flame, minimum three standards and the readings
were recorded. The recorded readings were used to prepare a calibration curve.
Determination of Total Amino Acids (Ishida et
al., 1981)


100mg of sample was weighed accurately into a heat
sealable test tube. 10ml of 6N HCl was added and the tube was heat sealed after
filling with pure nitrogen gas. Hydrolysis was carried out in a hot air oven at
110oC for 24h. After hydrolysis, the content was removed
quantitatively and filtered into a round bottom flask through whatman filter
paper No.42. The content of the flask was flash evaporated to remove traces of
HCl and the process was repeated 2-3 times with added distilled water. The
residue was made up to 10ml with C buffer (sodium citrate tribasic, perchloric
acid, n-caprylic acid, Ph 2.2). The sample was prepared and filtered again
through a membrane filter of 0.45µ for HPLC analysis.


30µl of the final filtrate was injected into Shimadzu
HPLC-LC10AS consisting of column packed with a strongly acidic cation exchange
resin i.e styrene di-vinyl benzene copolymer with sulfonic group. The column is
Na type i.e ISC-07/S1504 Na with a length of 19cm and 5mm diameter. The mobile
phase of the system consisted of two buffers, Buffer A (Tri sodium citrate
32.7g, Methanol of 140m, perchloric acid 16.6ml Ph 3.2 make up to 2 liter) and
buffer B (Tri sodium citrate 117.6g, Boric acid 24.8g, 4N NaoH 45ml, Ph 10).
The oven temperature was maintained at 600C. The amino acids were
eluted from the column by stepwise elution i.e acidic amino acids first
followed by neutral and the basic amino acids. The amino acid analysis was done
with non-switching flow method and fluorescence detection after post-column
derivatisation with O-phthaldehyde in the case of proline and hydroxyl proline,
imino group was converted to amino group with sodium hypochlorite. Amino acid
standards (Sigma Chemical Co., St. Louis, USA) were also run to calculate the
concentration of amino acids in the sample. Calibration of equipment using
standards was done before the start of analysis.

Quantification of amino acids: the standard and the
sample were analysed under identical conditions. The elution time of the amino
acids of the sample was compared and identified with those of the standard.
Quantification of amino acids was done by comparing the respective peak areas
in the chromatogram of the sample and standard. The amino acid content was
calculated as follows:

mg amino acid/g tissue = µmol*mol.wt*volume made

of sample

The amount of each amino acid was expressed as mg
amino acid/g tissue Estimation of tryptophan (Sastry and Tummuru,

300mg of sample was hydrolysed with 10ml of 5% NaOH at
110oC for 24hours in a sealed test tube filled with pure nitrogen.
The hydrolysate was neutralised to pH 7.0 with 6N HCl using phenolphthalein as
indicator and checked with Ph paper. The volume was made up to 100ml with
distilled water. This was then filtered through whatman filter paper No. 1 and
the filtrate was used for estimation. 0.1ml of 2.5% sucrose and 0.1ml of 0.6%
thioglycolic acid were added to the test tube containing 4ml of 50% H2SO4.
These was vortexed for 1 minute for proper mixing and kept for
5minutes in water bath at 45-50oC and cooled. An aliquot of the
sample was then added to the test tubes at different concentration. The
experiment was repeated with 0.1 to 0.8ml of standard tryptophan (10µg/ml). The
volume of the mixture in the test tubes was made up to 5ml with 0.1N HCl and
allowed to stand for 5 minutes for colour development. The optical density (OD)
was measured against a reagent blank at 500nm. Lipid profiling (Folch, et.al. 1957) Saponification and
Separation of Non-Saponifiable Matter (NSM)

Lipid was extracted from the fish muscle using Folch et al 1957 method by homogenising 30g
of the minced sample with 150ml of chloroform methanol (2:1). The mixture was
filtered using a Buckner funnel with Whatman No. 1 filter paper applying a
little vacuum. The extraction and filtration was repeated three times with the
residue. The three extracts was pooled together, transferred to a separating
funnel, 20% of the volume water was added, well mixed and left overnight to
allow separation. Lower layer was filtered through anhydrous sodium sulphate,
flash evaporated and concentrated to 10ml lipid with chloroform. 1ml of the
aliquot was taken into a pre-weighed vial which was kept for evaporation in an
oven. Vial was cooled in a desiccator and weighed. Fat content was calculated

Fat content (g/100g meat) = W2 x V1
x 100

                                                 V2 x W1

Appropriate volume of lipid containing 2g of fat was
taken and solvent was evaporated off. 30ml methanol and 1.5ml of KOH was added
and refluxed for 30 minutes in a boiling water bath under nitrogen. The
solution was cooled and transferred to a separating funnel while 20ml of
distilled water was added to it. The solution was extracted three times with
petroleum ether.
Extraction of Fatty Acids and Preparation of Fatty Acid Methyl Ether (FAME)

Fatty Acid Methyl Ether was prepared by acidifying the
aqueous layer from the previous step with concentrated HCl and checked with
litmus paper. The acidified layer was extracted three times with petroleum
ether and washed three times with distilled water. The washed extract was flash
evaporated to remove solvent. 6ml of BF3-methanol was added to the
flask after evaporation and refluxed in a boiling water bath for 6 minutes. It
was cooled and 6ml saturated NaCl was added after which it was transferred into
a separating funnel for further extraction. Extraction was done three times
with petroleum ether and washing of the extract carried out three times with
distilled water after which it was filtered through anhydrous sodium sulphate.
The filtered extract was flash evaporated and volume was made up to 1ml with
petroleum ether for Gas Chromatography analysis. Determination of pH (AOAC, 2000)

5g of the sample was homogenised in a beaker with 10ml
distilled water and the pH was measured using a digital pH meter and recorded. Determination of Thio Barbituric acid (TBA)

stability of the samples were assessed as Thiobarbituric Acid (TBA) value
spectrophotometrically by the modified method of Tarladgis,Watts, and Younathan  (1960). 10g of the sample was macerated with 100ml 0.2N HCl
and made into a slurry. Slurry was transferred into a round bottom flask and
connected to a TBA distillation apparatus. The slurry was distilled by steam
distillation method and 50ml of the distillate was collected. TBA reagent was prepared
by weighing 0.288gms of TBA standard and dissolving it in 100ml glacial acetic
acid in hot water bath and cooled to room temperature. 5ml of the distilled
sample was taken in test tubes and 5ml of the prepared TBA reagent was added. A
blank was made with 5ml reagent and 5ml distilled water in place of the
distilled sample. Then the samples were kept in boiling water bath for 30
minutes for colour development. The optical density (OD) was determined at
538nm wavelengths against blank in a spectrophotometer. The TBA value is
expressed as mg malonaldehyde/kg of fish.
Total Volatile Base Nitrogen (Conway, 1950)

Total volatile base in the sample was determined as
total volatile base nitrogen (TVB-N) by the micro diffusion method. One ml of standard
N/100 sulphuric acid was taken in the inner chamber of the diffusion unit. To
the outer chamber, 1ml of TCA extract was added followed by 1ml of saturated
potassium carbonate. Reagent blank was prepared using TCA solution in place of
TCA extract. The units were left overnight and the acid in the central chamber
was titrated against 0.01N NaOH using a drop of Tashiros indicator.
Tri methyl Amine (Conway, 1950)

Tri methyl amine is determined by the micro diffusion
method. One ml of standard N/100 sulphuric acid was taken in the inner chamber
of the diffusion unit. To the outer chamber, 1ml of TCA extract was added,
followed by 1ml of formaldehyde and 1ml of saturated potassium carbonate.
Reagent blank was prepared using TCA solution in place of TCA extract. The
units were left overnight. TMA absorbed in the acid in the central chamber is
estimated by titrating against 0.01N NaOH using a drop of Tashiros indicator.
Determination of Free Fatty Acids (AOAC, 1989)

10g of the sample was blended with anhydrous Na2SO4
in a mortar. The blend was shaken with chloroform and kept in dark overnight
and filtered. 20ml of the extract was taken into a pre-weighed clean beaker and
put in a water bath for chloroform to be evaporated off. This was subsequently
dried off in hot oven and cooled in a desiccator. Weight of fat was determined
by subtracting the initial weight of the flask from the final weight containing
the fat extract was transferred into a conical flask and the chloroform was
evaporated off. 10ml of neutral alcohol was added and it was warmed in a water
bath. Reagent blank was done using chloroform in place of chloroform extract,
to get the blank value. It was titrated against 0.01N NaOH using
phenolphthalein as indicator. Percentage of free fatty acid (FFA) was
calculated as oleic acid.  Percentage of
free fatty acid (FFA) was expressed as % oleic acid.
Instrumental Colour Determination 

Hunter Lab MiniScan® XE Plus spectro-colorimeter, model No D/8-S (Hunter
Associates Laboratory lnc., Reston, VA, USA) with geometry of diffuse 80
(Sphere-8 mm view) and an illuminant of D 65 optical sensor and100 standard
observer was used for instrumental colour measurement of the samples. The
homogenised sample was filled into the sample holder made of glass (2.5 inch
round), fitted into the grove provided for the colour analysis. The colour
values were expressed using the standard CIE L*a*b* system. L*, a*, and b*
values refer to the three axes of the system: a lightness L* and two axes
representing hue and chroma, (a*) and the other (b*) blue-yellow. The colour instrument
was first standardised with a white plate (Illuminant C: Y = 93.6, x = 0.3134,
and y = 0.3194). Three times of random measurements were taken from each sample.
The average of the three replicate measurements was used to calculate the
hue-angle (h) which represents the relative position of colour between redness and
yellowness and chroma (C*)
Determination of Texture Profile

The TPA method of Bourne (1978) based on compression
of samples with Universal Testing Machine (Lloyd instruments LRX plus) was used
to objectively evaluate textural differences between treatments. Uniform size
of the fish samples were used for the analysis. The load cell used was a
cylindrical probe of 50mm diameter with 50N capacity. The samples were
compressed twice to 40% of their original height at a crosshead speed of
12mm/min. Force by time data from each test were used to calculate mean values
for the TPA parameters. The values for hardness 1 and 2 (the resistance at
maximum compression during the 1st and 2nd compression to
that during the 1st compression of Area2/Area1), springiness (ratio
of the time duration of force input during the 2nd compression to
that during the 1st compression of length 2/length 1), and chewiness
(hardness 1 x cohesiveness x springiness in kg mm) were determined as described
by Bourne (1978). Compression was carried out triplicates and average readings
were taken.

3.3.10 Sensory Evaluation

sensory analysis was conducted according to the Quality Descriptive Analysis
(QDA) method. The 9-point hedonic scale was chosen as the test method. Ten
trained panelists were used for the sensory analyses. The sensory attributes
evaluated were appearance, texture, taste, flavour, chewability and overall
acceptability. Portable water was provided for rinsing mouth after tasting each
sample, to minimise error and avoid masking perception of the attributes.  Questionnaire was prepared based on the
9-point hedonic scale where 9 represented extremely acceptable, 8- very
acceptable, 7- moderately acceptable, 6- slightly acceptable, 5- neither
acceptable nor unacceptable, 4- slightly unacceptable, 3- moderately
unacceptable, 2- very unacceptable and 1 represented extremely unacceptable.
The obtained values were subjected to statistical analysis using Analysis of
Variance (ANOVA) to determine the significance difference between the samples.

(ANOVA) was performed using SPSS
(Software Package for Social Scientists) version 15.0.



3.4 Sterility Test (IS: 2168-1871)

The thermally processed samples were tested for
commercial sterility. Four pouches were randomly selected from each sample, two
pouches were incubated at 37oC for 14 days and the other two at accelerated
temperature of 55oC for 4 days. The incubated pouches were
aseptically opened and 1-2g of the samples were taken by a sterilised forceps
and inoculated into sterile thioglycolate broth in test tubes. A layer of
sterile liquid paraffin wax was put on top of the broth to create anaerobic
condition and the tubes were incubated. Development of turbidity
which indicates growth and survival of microorganisms was looked out for. Tubes
which did not show any turbidity confirmed the sterility of the samples.

Storage Studies

The retort pouched products were stored under room
temperature (28 ± 2oC). Sensory evaluation, proximate analysis as
well as biochemical analyses were conducted at the end of every month. The
keeping quality studies lasted for a period of twelve months.

3.6 Statistical Analysis (XLSTAT Pro 7.5)

All the data obtained were subjected to statistical
analysis using descriptive
statistics and ANOVA at ?-0.05. Data were expressed as mean ± standard deviation. Multiple
comparison tests were performed for each of the variable involved in the
processing. The differences between the means of samples were evaluated using
Fisher’s F test at 95% confidence level. Therefore,
differences were considered to be significant at p<0.05.