III. MATERIAL AND METHODS In the present study, fresh octopus (Octopus membranaceus) was obtained from

Mangalore landing centre in iced condition. Different batches of octopus caught in

different trawls collected from Mangalore landing centre were used for different

processing and storage operations viz., chilling, smoking and preparation of battered and

breaded and frozen products. Raw material quality was assessed by physical, chemical,

microbiological and sensory tests. Preliminary studies such as method of dressing,

chemicals treatment, battering & breading and smoking operations were standrdised.

The dressed raw material was divided in to different batches, one batch of whole

octopus, one batch of dressed octopus and one batch of dressed, packed and sealed in

poly ethylene bags were used for chilled storage studies using ice.

One batch of dressed octopus was used for the development of battered and

breaded octopus. Another batch of dressed octopus was used for the development of

smoked octopus. After preparation both the products were frozen in air blast freezer at -

35 oC and stored at -20 oC.

Another four batches of dressed octopuses were used for cryoprotectants

treatments such as sodium chloride, sodium tri-polyphosphate, sodium citrate, ascorbic

acid (individually or in combination) to reduce the drip loss during thawing and hydrogen

peroxide to improve the meat colour. One batch of dressed octopus was kept as control.

All the batches were individually quick frozen in an Air blast freezer at -35 oC and stored

at -20 oC. The quality characteristics of the stored products were assessed by physical,

chemical and sensory tests at regular intervals. The materials used and the methods

followed are described in this section.

3.1. Materials; 3.1.1. Raw material

Octopus which are caught in the deep sea trawl and kept on board the vessel in

chilled condition using ice in a fishing trip of 3-5 days were collected from the local

landing centre immediately after reaching the landing centre. The octopuses were in post

rigor condition. They were brought to the processing hall in iced condition and processed

with in 8 hours.

3.1.2. Packaging material Low density polyethylene (LDPE) bags of 500 g capacity were used for

packaging of dressed materials and the finished products and were heat sealed. Three

ply master cartons were used for storing the frozen products.

3.1.3. Ice box

Insulated ice box SKCFMF designed by MPEDA was used for ice storage

studies.

3.1.4. Ice

Good quality block ice was obtained from the local ice plant and crushed in ice

crusher and used for ice storage studies.

3.1.5. Saw dust and wood shavings

Saw dust from hard wood (teak, mango, jack) were obtained from a local saw mill

for use in the smoking process.

3.2. Sub materials a) Seasoning mixture

Commercially available “Captain cook” iodised / refined table salt, Everest brand

chilly powder, Priya brand ginger paste and garlic paste were used.

b) Battering mixture Commercially available refined wheat flour (maida) was used as predusting

material after mixing with seasoning mixture and for preparation of battering dough,

same maidha, salt and potable water were used.

c) Bread crumbs Locally produced / available bread powder from a bakery was sieved and used

for breading of battered octopus.

d) Vegetable oil Refined groundnut oil (Dhara brand) was used for frying the battered and breaded

product.

3.3. Equipments Protein sample digestion system, distillation unit, muffle furnace, hot air oven,

incubator, autoclave, mixie, tissue homogenizer, pH meter, electronic balance,

spectrophotometer, electric heat sealer, electric hot plate, electric boiler / water bath,

refrigerator, Remi-24 centrifuge etc. were used to carry out different operations and

analyses.

3.3.1. ABS - Air blast freezer Prepared products were quick frozen using ABS - air blast freezer at -35 oC.

3.3.2. AFOS Torry mini kiln

Smoking was carried out using an AFOS Torry mini kiln pilot plant charged with

shavings of hard wood and saw dust. The quantity of smoke entering the chamber was

regulated by lighting one or two saw dust chambers. The kiln is essentially a wind tunnel

in to which smoke is drawn by a centrifugal fan from the smoke producer facilities the

flow of smoke over the trolley where octopuses are kept / hung to the stainless steel rods

and a controllable proportion of the moisture laden smoke is then vented up to a

chimney. Fresh air and smoke are continuously introduced in to the kiln and appropriate

temperature is maintained by thermostatically controlled electric heater. Uniform smoke

flow is obtained by fixed baffles and plates.

3.4. Chemicals Most of the chemicals used were either from SQ special quality laboratory

reagents, Qualigens fine chemicals, Bombay and Ranbaxy laboratory Ltd., Punjab.

3.5. Microbiological media and reagents Culture media required for TPC estimation, Salmonella spp. and Vibrio spp.

enumeration were obtained from Hi-Media (Hindustan dehydrated media), Bombay.

3.6. Methods;

3.6.1. Study of raw material characteristics

Physical, chemical, sensory and microbiological qualities of raw and dressed

octopuses were analysed.

3.6.1.1. Physical characteristics

Physical characteristics of the octopuses were analysed by measuring total

length, dorsal mantle length (DML), tentacle length (Fig.1), total weight of randomly

selected 50 specimens in a batch and yield of dressed octopuses were calculated as

percentage of cleaned octopus weight over the raw octopus weight.

3.6.1.2. Sensory characteristics Fresh octopus was analysed for sensory characteristics like appearance, odour,

texture, flavour and overall acceptability using 9 point hedonic scale. The proforma used

for sensory evaluation is presented in appendix-I.

3.7. Pre-process operation / Processing method

The raw material was cleaned by cutting the tentacles just behind the eyes and

separated the mantle. Eyes were removed from the tentacle part, then skin is pulled from

the cut end and peeled, suckers and small portion of the tentacles were trimmed off by

knife. The mouth contains beaks which are also removed. Then skin of the mantle was

removed by pulling / peeling it from the cut end of the mantle. By turning the inside of the

mantle out, internal organs such as gills, branchial heart, kidney, gonad, pancreas, liver,

ink sac etc. were removed and washed thoroughly.

3.8. Ice storage studies

One batch of whole octopuses were directly iced without any dressing (WOI), one

batch of dressed octopuses were directly iced (DOI) separately in insulated ice boxes in

alternative layers of ice and octopus in 1:1 ratio. The another batch of dressed octopus

was packed in LDPE bags and heat sealed to avoid direct contact with ice and iced (DPI)

in alternative layers of octopus and ice in 1:1 ratio. Sufficient ice was covered on the top

to avoid exposure to atmosphere temperature and lid of the insulated box was kept

closed tightly and stored at ambient conditions. During storage, melted water was

removed and replenished with fresh ice. The process of Icing of octopus is given in flow

chart (Fig. 2) and plate (Fig. 3). The stored octopuses were sampled in triplicate once in

three days to evaluate the changes in quality during storage.

3.9. Production of battered and breaded octopus (BBO) The battered and breaded octopus was prepared from the dressed octopus. The

procedure followed is given in flow chart (Fig. 4) and plate (Fig. 5). Dressed and iced

octopus was mixed with the spices mixture (Table 2) and allowed to stand for 30 min. at

room temperature. Then they were pre dusted using maidha flour. The pre dusted

material was dipped in a batter solution.

3.9.1. Standardisation of spice mixture Spice mixture was formulated keeping in view of the food habits of the local

population. Four different spice mixtures were prepared using different combination of

seasoning mixtures viz.,

1. Chilly powder (3%) and salt (2%)

2. Chilly powder (3%), tamarind paste (1%) and salt (2%)

3. Chilly powder (3%), tamarind paste (1%), onion paste (2%) and salt (2%)

4. Chilly powder (2%), ginger (1%), garlic (1%) and salt (2%).

After mixing the seasoning mixture with the dressed material, they were allowed to

stand for 30 min. Then they were predusted using maidha flour. Then they were battered

using maidha, salt and water of desirable consistency and rolled over bread crumbs. The

prepared products were then fried in refined vegetable oil and presented to minimum 10

trained panelists for sensory evaluation.

3.9.1.1. Standardisation of batter Commercially available maida flour and rice flour were selected for preliminary

standardization studies. Maintenance of constant batter viscosity is important for batter

preparation. Considering the water absorption capacity and viscosity, batter of different

viscosity were tried and standardized. Maida batter solution was standardised in different

solutions having different viscosity. The water absorbed by known quantity of maidha

was calculated by noting down the amount of water used to prepare batter of desired

viscosity to get uniform coating. The quantity of water required to prepare a batter of

desired consistency was fixed by measuring the viscosity of batter which should be

uniform. In the elaborate apparatus, the viscosity of batter solution was measured by

noting down the time for certain quantity of batter at room temperature to flow through a

small orifice of a funnel (Holston, 1956) (dia. 5 mm and length 9.5 cm). The diameter of

the funnel used was 10 cm.

3.9.2. Breading The battered octopuses were rolled over bread crumbs. The change in weight of

meat during mixing and coating was measured. To calculate the weight change during

each step, 25 mantle and tentacle pieces were selected at random and the weights were

noted down to calculate the percentage of picking during mixing and predusting,

battering and breading steps.

3.9.3. Freezing

The battered and breaded octopus was frozen individually in an air blast freezer

at -35 oC and stored at -20 oC. The stored product was sampled in triplicate once in a

month to evaluate the changes in quality during storage.

3.9.4. Frying

The frozen product after thawing were deep fried in refined groundnut oil at 180-

200 oC for 3 to 5 min. to a golden brown colour by traditional open pan frying technique

for sensory evaluation.

3.10. Production of smoked octopus (SMK)

The smoked octopus was prepared from the dressed octopus. The procedure

followed is given in flow chart (Fig. 6) and plate (Fig. 7) dressed octopus was brined and

arranged in kiln in such a way that the surface area was exposed to the smoke to the

maximum extent and smoked for standardized time and temperature.

3.10.1. Standardisation of salt concentration / brining for smoking The dressed octopus were divided in to 3 batches and each batch was dipped in

6%, 8%,10% and 12% NaCl solution for 4 min. after which the octopus was rinsed in

fresh water to remove excess of surface salt and subjected to smoking.

3.10.2. Standardisation of smoking time After 10% salt treatment for 4 min., the octopus meat was subjected to different

smoking times (75, 90, 105, 120 and 135 min.) at 80 and 90 oC and then they were

subjected to panel test. Based on the organoleptic panel tests, a suitable smoking time

was selected for further experiment.

3.10.3. Packing Smoked product was packed in LDPE bags and frozen at -35 oC. The frozen

smoked product was stored at -20 oC in cold storage. The battered and breaded octopus

product was frozen individually in an air blast freezer at -35 oC and stored at -20 oC. The

stored product was sampled in triplicate once in a month to evaluate the changes in

quality during storage.

3.11. Treatment with cryoprotectants / chemicals Dressed octopus was used for treatment with cryoprotectants / chemicals to

improve the frozen storage stability and appearance of dressed octopus.

3.11.1. Standardisation of concentration and time for pretreatment The concentration of cryoprotectants / chemical and time of dip treatment was

standardised.

3.11.2. Standardisation of sodium tri-poly phosphate treatment

After dressing, dressed material was divided in to four batches and dipped in to

2%, 3%, 4% and 5% STPP solution for 5 min. and frozen at -35 oC and stored at -20 oC

in cold storage. Samples were drawn once in a month to analyse for drip and sensory

evaluation.

3.11.3. Standardisation of sodium citrate Dressed octopus material was divided in to four batches and each batch was

given dip treatment in 1%, 2%, 3% and 4% sodium citrate solution for 5 min. and frozen

at -35 oC and stored at -20 oC in cold storage. Samples were drawn once in a month to

analyse for drip and sensory evaluation.

After standardisation of concentration for STPP and SC treatment, dressed

material was divided in to 4 batches. First batch was dipped in 5% salt (sodium chloride)

solution (Fig. 8) for 5 min. then in mixture of solutions of 4% sodium tri-poly phosphate and 0.1% ascorbic acid (AC) for 5 min. (for every one kilogram of dressed octopus two

liters of treatment solution was used in all the treatments) = STPP.

The second batch was first dipped in 5% salt (sodium chloride) solution for 5 min.

then in mixture of solutions of 3% sodium citrate and 0.1% ascorbic acid for 5 min. = SC.

The third batch of the dressed octopus was dipped in 1% hydrogen peroxide

solution for 15 min. = H2O2. The aim of dipping in hydrogen peroxide is to improve the

colour of the meat.

The last batch of the dressed octopus was untreated and kept as control (CTL).

After giving treatment all the batches including control were packed separately in LDPE

bags and frozen at -35 oC and stored at -20 oC in cold storage. The stored samples were

drawn once in a month in triplicates to analyse the changes in quality by biochemical,

microbiological and sensory methods.

3.12. Evaluation of quality changes during storage: The ice stored octopus samples were analysed for bio-chemical and

microbiological quality parameters such as moisture, protein, total lipids, ash, total

volatile base nitrogen (TVBN), Trimethylamine nitrogen (TMAN), peroxide value (PV),

free fatty acids (FFA), Hypoxanthine (Hx), pH, salt soluble nitrogen (SSN), water soluble

nitrogen (WSN), non protein nitrogen (NPN), total plate count (TPC), Psychrophiles and

sensory evaluation.

Frozen stored battered and breaded octopus was analysed with coating for

moisture, TPC, Salmonella spp., Vibrio cholerae, Vibrio parahaemolyticus and sensory

evaluation and without coating for total protein, crude fat, SSN, NPN, TVBN, alpha amino

nitrogen (AAN), PV, FFA and pH. Smoked octopus was analysed for all the above

parameter in addition to steam volatile and non volatile phenolic compounds.

Pretreated octopuses were analysed for moisture, total protein, total lipids, SSN,

NPN, TVBN, AAN, PV, FFA, pH, drip loss, cook loss, SDS-PAGE, TPC, Salmonella spp.

Vibrio cholerae and Vibrio parahaemolyticus to assess the changes in quality during

storage.

3.12.1. Biochemical analysis 3.12.1.1. Estimation of phosphorus Estimation of phosphorus was done by following the procedure described in ISI

specifications for poultry feed. Ashed sample was boiled with 40 ml of HCl (25 ml of

concentrated HCl in 100ml distilled water) and filtered through a dry filter No.44

Whatman, the filter paper was washed 2 - 3 times with hot water and made up to 100ml.

10 ml of the made up solution was taken in a beaker. In another separate beaker, 10 ml

con. HNO3 was taken, to that 10 ml of ammonium molybdate stock solution (200g of

Ammonium molybdate was taken in stoppered graduated cylinder of 1000 ml capacity,

800 ml of water was added and shook well for 25 min. to dissolve, gradually 25% (w/v)

ammonium hydroxide solution was added till the solution turns clear, volume was made

up to 1 litre, this may be filter if necessary.) was added whirling the beaker during

addition as this was poured in to the beaker containing 10 ml of filtered ash solution,

stirred and allowed for precipitate for 15 min. Then it is filtered through Whatman filter

paper. The filter paper was washed with dilute HNO3 and potassium nitrate solution until

it is free from acid.

For titration, the precipitate with filter paper was transferred to beaker in which

precipitation was carried out. Sufficient volume of standard NaOH was added from the

burette (just sufficient to dissolve the precipitate) and then add 5 ml extra to dissolve

precipitate (yellow) completely. Noted the volume of standard NaOH added. Then 10

drops of phenolphthalein indicator was added and excess alkali was titrated against

standard HNO3.

Calculation: 1 ml of 0.1 N NaOH = 0.000304 g of P2O5

(V1N1 – V2 N2) x 0.000304 x 100 x 100

P2O5 (%) = ------------------------------------------------------ W x 10 Phosphorus % = P2O5 x 0.4365

Where, V1 = Volume of NaOH, N1= Normality of NaOH, V2 = Volume of HNO3,

N2 = normality of HNO3, 10/100 = dilution ratio, 100 = %, W = sample weight.

3.12.1.2. Moisture Moisture content of the sample was determined by following the hot air oven

method (AOAC, 1995). 5-10 g of the octopus meat sample was taken in a moisture

bottle, dried in a hot air oven maintained at 100 5 oC for 16-18 hours and cooled in a

desiccator. The sample was repeatedly heated, cooled and weighed till a constant

weight was observed. The weight loss during this process was expressed as the

moisture per 100 g of sample.

3.12.1.3. Total protein

Estimation of total nitrogen in the octopus product was done using micro Kjeldahl

method (AOAC, 1995). The nitrogen in the sample was converted in to ammonium

sulphate by digesting the sample with concentrated sulphuric acid. Ammonium sulphate

formed was decomposed by alkali and the liberated ammonia absorbed in 2% boric acid

and titrated against standard (0.05 N) Sulphuric acid. The protein value is calculated by

multiplying the nitrogen content by 6.25.

3.12.1.4. Ash Moisture free samples were incinerated and ashed in a muffle furnace at a

temperature of 550 - 600 oC for 4-5 hours (AOAC, 1995) and the values were expressed

on wet weight basis as percentage.

3.12.1.5. Total lipids

Total lipid was estimated by the method described by Bligh and Dyer (1959). 20 g

of the minced meat was homogenized with mixture of 40 ml methanol and 20 ml of

chloroform for 2 min. Then 20 ml of chloroform was added and homogenized for 30

seconds after adding 20 ml of distilled water. The resulting homogenate was transferred

to Buchner funnel and filtered using suction pressure. The filtrate was transferred to

separating funnel and heavier chloroform layer was separated. An aliquot of 5 ml

chloroform extract was dried in a vacuum oven. The percentage of total lipid in the

sample was calculated as follows.

Weight of lipid in an aliquot x total vol. of Chloroform (ml) Total lipids = --------------------------------------------------------------------------- x 100 Vol. of aliquot (ml) taken x weight of sample taken (g)

3.13. Lipid quality; 3.13.1. Peroxide Value (PV)

Peroxide value of extracted fat was determined by titrimetric method of Tarr

(1947). 5 ml of the chloroform extract was mixed with 25 ml of PV solvent (chloroform

and glacial acetic acid in 2: 1 ratio), 1 ml of saturated potassium iodide and the liberated

iodine was titrated against standard sodium thiosulphate solution using starch as an

indicator and expressed as millimoles of peroxide oxygen / kg of fat.

1000 x (Titre value of sample- TV of blank) x N of Na2 S2 O3 PV (millimoles of O2) = ------------------------------------------------------------------------------- Weight of fat taken 3.13.2. Free fatty acids (FFA) Free fatty acid was determined by the method described Olley and Lovern

(1960). 5 ml of the chloroform extract was evaporated on water bath, dissolved in 50 ml

of hot neutral alcohol and titrated against standard alkali and expressed as oleic acid

percent in total lipids.

28.2 x (Titre value of sample- TV of blank) x N of NaOH FFA (% of oleic acid) = --------------------------------------------------------------------------- Weight of fat taken 3.14. Freshness indices

Total volatile base nitrogen (TVB-N) and Tri-methylamine nitrogen (TMA-N): were

estimated for freshness indices.

3.14.1. Preparation of TCA extract

About 10g of sample was ground well with 20 ml of 20% TCA. The homogenate

was filtered through Whatman No.1 filter paper and the filtrate was made up to 100ml

with distilled water.

3.14.2. Total volatile base nitrogen (TVB-N)

1ml of the extract was put in outer chamber of Convey’s unit and 2ml of boric acid

in the inner chamber. Partially lid was closed and 1 ml of saturated potassium iodide was

added to the outer chamber and lid was closed and incubated at 37 oC for 90 min. After

incubation, the inner chamber was titrated against standard H2SO4 solution. Blank was

carried out using 2% TCA instead of sample (Beatty and Gibbons, 1937).

14.01 x N of H2SO4 x (Titre value of sample-TV of blank) x 100 x 100 TVB-N = --------------------------------------------------------------------------------------------- x1000 (mg %) 1000 x weight of sample

3.14.3. Trimethylamine-Nitrogen (TMA-N) TMA-N content of the octopus samples was estimated by the method of Beatty

and Gibbons (1937) using Convey’s micro diffusion technique. 1ml of the extract was

taken in outer chamber and 2 ml of 2% boric acid containing mixed indicator was put in

inner chamber of the Convey’s unit and the lid was partially closed. 1 ml of formalin and

1 ml of saturated potassium carbonate was added to the outer chamber and the lid was

immediately closed and incubated at 37 oC for 90 min. After incubation, the inner

chamber was titrated against standard H2SO4 solution. Blank was carried out using 2%

TCA instead of sample.

14.01 x N of H2SO4 x (Titre value of sample-TV of blank) x 100 x 100 TMA-N = -------------------------------------------------------------------------------------------- (mg %) weight of sample x 1

3.14.4. Alfa amino nitrogen (AAN) AAN was estimated by the method of Pope and Stevens (1939). 5 g of the meat

sample was ground thoroughly with 10 ml of 20% TCA and filtered. The filtrate was

made up to 100 ml in volumetric flask. 10 ml of extract was taken in 50 ml vol. flask and

few drops of thymolphthlein was added and the extract was made alkaline (blue colour)

using Normal NaOH. 30 ml of mixture of solutions of (1 part copper chloride and 2 parts

of trisodium phosphate and 2 parts of borate buffer by volume) was added and the

volume was made up to 50 ml with distilled water. After shaking and allowing it for 15

min. filtered. 10 ml of filtrate was taken in a conical flask, 0.5 ml of glacial acetic acid and

0.5 g of potassium iodide was added. The liberated iodine was titrated against N/500

sodium thiosulphate using starch as an indicator (as in PV estimation).

1 ml of 0.01N Na2S2O3 = 0.28 mg of AAN

Titre value of sample x 0.28 x 100 x 50 X 100 AAN (mg %) = ------------------------------------------------------------- Weight of sample x 10 x 10

3.14.5. Salt soluble nitrogen (SSN) Salt soluble nitrogen was extracted by the method of Dyer et al., (1950). Ten

grams of octopus meat was homogenized with 200 ml of chilled 5% NaCl solution

buffered with 0.02 M NaHCO3 and pH adjusted to 7.0 to 7.5 by using 0.1 N HC1. The

total volume of the homogenate was made upto 250 ml and centrifuged at 6000 rpm for

10 min. and 10 ml of supernatant was made up to 100ml, from this 5 ml was taken for

nitrogen estimation using micro Kjeldahl method. The SSN was calculated and

expressed as milli gram percent.

14.01 x N of H2SO4 x (TV sample-TV blank) x 250 x 100 x 100 SSN (mg %) = -----------------------------------------------------------------------------------

10 x 10 x weight of sample

3.14.6. Non protein nitrogen (NPN) 5 g of the octopus meat was ground well with pestle and mortar, the slurry was

extracted with 50 ml distilled water. Then 10 ml of 20% TCA solution was added to

precipitate the proteins. The solution was filtered and filtrate was made up to 100 ml

using a volumetric flask. 5 ml of an aliquot was used to determine the nitrogen content by

micro Kjeldahl method.

14.01 x N of H2SO4 x (TV sample-TV blank) x 100 x 100 NPN (mg %) = --------------------------------------------------------------------------

5 x 5 x Vol. taken for distillation

3.14.7. Water soluble nitrogen (WSN) 5 g of octopus meat was mixed with 95 ml of distilled water, homogenized and

filtered. The filtrated homogenate was made up to 100 ml. The nitrogen content in an

aliquot (5 ml) taken was determined using micro Kjeldahl method.

14.01 x N of H2SO4 x (TV sample-TV blank) x 100 x 1000 WSN (mg %) = ----------------------------------------------------------------------------

5 x 5 x Vol. taken for distillation

3.14.8. pH 5 g of octopus minced meat was homogenized with 45 ml of distilled water and

was transferred to sterile glass beaker and kept in refrigerator for 30 min. The pH of the

homogenate was measured with the help of combined electrode pH meter after

calibration with standard pH. 4.2 and 9.0 buffer solutions.

3.14.9. Steam volatile and non volatile phenolic compounds

Total phenol content in the smoked octopus meat was estimated according to the

method of Foster and Simpson (1961). Twenty grams of the sample was ground with

160 ml of distilled water and the resulting mixture was transferred to one litre flask with

the addition of 40 ml distilled water. The sample was steam distilled and the liquid level

in the flask during distillation was kept constant by a constant supply of steam. After the

collection of six 100 ml fractions, the steam supply was shut off and the steam volatile

phenol content of the fraction was determined. The residue in the distillation flask was

cooled and diluted to make 250 ml with distilled water before the steam non-volatile

phenol content was determined. The steam volatile and steam non-volatile phenolic

contents were determined as follows.

An appropriate volume of the aqueous solution of phenols was pipetted out into a

25 ml standard flask and made alkaline with aqueous sodium carbonate solution (3 ml,

2% w/v) and a freshly made aqueous solution of 4-aminoantipyerene (2 ml, 1% w/v).

After dilution of the mixture, one ml of potassium ferricyanide solution (8% w/v) was

added and contents of the flask were thoroughly mixed. The colour intensity was

measured at 550 nm using Bausch and Lomb Spectronic 20. The results were

expressed as mg%. The standard graph was drawn using standard phenol solution (Fig.

9).

3.14.10. Hypoxanthine (Hx) 5.0 ± 0.05 g of octopus muscle free from skin blood was blend with 50 ml of 0.6M

perchloric acid and Filtered. To the 5.0 ml of filtrate, 5.0 ml of potassium hydroxide

phosphate buffer was added, chilled and filtered (Kept this extract chilled, when the

analysis was not able to complete on the same day, stored in freezer) (Jones et al.,

1964).

Preparation of reaction mixture: 0.5, 1.0 or 2.0 ml of filtrate (depending on the

anticipated hypoxanthine content) was taken in to a test tube and made up to 2.0 ml with

water. 1ml of filtrate is sufficient for most of the analyses. 2.0 ml of 0.25 M phosphate /

EDTA buffer and l.0 ml of enzyme working solutions were added. This is solution A.

Preparation of extract blank; For each extract pipetted the same volume of

extract as was used for A in to a test tube and made up to 2.0 ml as before, 2.0 ml of

0.25 M buffer and 1.0 ml of water was added, this is solution B.

Preparation of enzyme blank; For each set of determinations a blank of 2.0 ml

of water, 2.0ml of buffer and 1.0ml of enzyme working solutions were added. This is

solution C.

Preparation of buffer blank; For each set of determinations a blank containing

2.0 ml of buffer and 3.0 ml of water was prepared. All the mixtures were Incubated at

37 0C for 30 min. The absorbance was read at 293 nm using a 10 mm silica cell with

water in the reference cell. The net absorbance due to uric acid was calculated after

correcting for the blank as follows:

Abs = (A – D) – (B – D) – (C – D) = (A – B) – (C – D)

Calculation of hypoxanthine concentration; 1 The molar extinction coefficient of uric acid at 293 nm in a 10mm cell is 12.0 x

103. One mole of uric acid is equivalent to one mole of hypoxanthine, molecular weight

136.1. Therefore the amount of hypoxanthine in 5 ml of the reaction mixture is:

Abs x 136.1 x 5 = Abs x 0.0567 mg

12.0 x 103

3.14.11. Drip Loss Drip loss of the octopus meat was determined as follows; 20 – 30 g of frozen

sample was thawed in polyethylene bag at refrigerated temperature (2-5 °C) for 4-6

hours. After thawing, the liquid was removed by blotting with filter paper. The final weight

of sample was recorded and the result was expressed as percentage of initial weight.

Initial weight before thawing - Weight of sample after thawing Drip Loss (%) = ----------------------------------------------------------------------------------- x 100

Weight of sample taken 3.14.12. Cook Loss

Cook loss of octopus was determined by following the method of Kondaiah et al.,

method. 20 g of thawed sample was placed in a polyethylene bag and heated at 80 °C

for 20 min. in a water bath and drained out the exudate, the loss in weight is calculated

as a % of initial weight.

Initial weight before cooking - Weight of sample after cooking Cook Loss = ---------------------------------------------------------------------------------- x 100

Weight of sample taken 3.15. Total ammo acid analysis (Ishida et al, 1981)

The total amino acid analyses were carried out at the Department of Biochemistry

and Nutrition, Central Institute of Fisheries Technology, Cochin. About 100 mg of raw

octopus meat sample was weighed accurately into a heat sealable test tube. 10 ml of 6

N HCI was added and the tube was heat sealed after filling pure nitrogen gas. Hydrolysis

was carried out in a hot air oven at 110 °C for 24 hours. After the hydrolysis, the contents

were removed quantitatively and filtered into a round bottom flask through Whatman filter

paper No.42. The contents of the flask were flash evaporated to remove traces of HCI

and the process repeated for 2-3 times with added distilled water. The residue was made

up to 10 ml with 'C' buffer (sodium citrate tribasic, perchloric acid, n-caprylic acid,

pH.2.2).

The sample thus prepared was filtered again through a membrane filter of 0.45

µm and 20 µl of this was injected to Shimadzu HPLC-LC10AS consisting of column

packed with a strongly acidic cation exchange resin i.e. styrene divinyl benzene

copolymer with sulfinic group. The column is Na type i.e. ISC-07/S1504 Na with a length

of 19 cm and diameter 5 mm. The mobile phase of the system consists of two buffers,

Buffer A (sodium citrate, ethanol of 99.55%, perchloric acid 60%, pH 3.2) and buffer B

(sodium citrate, boric acid, 4N NaOH, pH 10.0). The oven temperature was maintained

at 60° C. The amino acids were eluted from the column by stepwise elution i.e. acidic

amino acids first followed by neutral and then basic amino acids. The amino acid

analysis was done with non-switching flow method and fluorescence detection after post-

column derivatization with O-phthalaldehyde. In the case of proline and hydroxy proline,

imino group was converted to amino group with sodium hypochlorite. Amino acid

standard was also run to calculate the concentration of sample amino acid depending on

the standard chromatogram. The results were quantified and represented as gram amino

acid per 16 g N2.

3.15.1. Estimation of tryptophan (Sastry and Tummuru, 1985) About 200 mg of raw octopus sample was hydrolysed with 10 ml of 5% NaOH at

110° C for 24 hours in a sealed tube filled with pure nitrogen. The hydrolysate was

neutralized to pH 7.0 with 6 N HCl using phenolphthalein indicator and checked with

BDH pH paper. The volume was made upto 100 ml with distilled water. This was then

filtered through Whatman filter paper No.1 and filtrate was used for estimation. 0.1 ml of

2-5% sucrose and 0.1 ml of 0.6% thioglycolic acid were added to test tube containing 4

ml of 50% H2SO4 and kept for 5 min. in water bath at 45-50 °C and cooled. An aliquot of

the sample was then added to the test tubes. The experiment was repeated with 0.1 to

0.8 ml of standard tryptophan (10 µg/ml). The volume was made upto 5 ml with 0.1 N

HCI and allowed to stand for 5 min. for the development of colour. The absorbance was

measured against a reagent blank at 500 nm in Spectronic20 Genesys.

3.16. Sodium dodecyI sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis

SDS-PAGE was performed according to the method of Laemmli (1970). The

protein extracts prepared were mixed with Laemmli sample buffer containing buffer, 1%

SDS, 25% glycerin, 2% mercaptoethanol and 0.2% bromophenol blue (pH 7.2), in the

ratio of 1:1 and boiled for 2 min. and kept frozen pending electrophoresis. The samples

(3µl) along with molecular weight marker (Sigma, USA) were loaded on to 4% stacking

gel and 12.5% separating gel and electrophoresed at 25 mA till the dye front reached the

bottom of the gel. The gel was stained with coomassie blue (10% acetic acid, 40%

methanol, 0.1% coomassie blue) for 5 h and destained with destaining solution (10%

acetic acid, 40% methanol). The electrophorogram was photographed and subsequently

scanned in computer.

3.17. Determination of heavy metals concentration The analysis of heavy metals concentration were carried out at the Department of

Biochemistry and Nutrition, Central Institute of Fisheries Technology, Cochin by following

the Shankar et al., 2006 method.

3.17.1. Preparation of samples An aliquot of the homogenized meat was made moisture free by pressing it with

in a filter paper. Then about 10-15g of the meat was weighed and transferred to a round

bottom flask. Then to it about 15ml of concentrated nitric acid was added and was kept

over night for predigestion. Duplicate samples were taken in all kind of analysis. In the

determination of heavy metals the organic matrix has to be completely oxidized.

Oxidation was carried out by wet digestion method using concentrated nitric acid and

concentrated perchloric acid in the ratio of 2:1 (v/v). Samples for determination of

mercury was digested in a Bethge's apparatus using concentrated nitric acid and

perchloric acid in the ratio 4:1 (v/v) under closed condition to prevent the escape of

volatile metals. During digestion it was cooled using a circulating water condenser.

Digestion was continued till a clear solution was obtained. The samples were cooled and

diluted to a known volume in a standard flask using MiliQ water of conductivity 18.2 µS.

A reagent blank was also prepared by taking the same volume of acid mixture and other

ingredient except the samples.

3.17.2. Preparation of Standards

The stock standard of metal was prepared from Analar grade metal salts and in

certain case standard metal solution of AAS grade obtained from Sigma chemicals Co.,

USA were used. The examples of preparation of some of the metallic standards have

been given here.

a) Zinc: 0.1 gram of pure Zn was dissolved in 2 ml hydrochloric acid and was diluted to

1000ml.

b) Lead: 0.1598 g of lead nitrite was dissolved in nitric acid, to this10ml nitric acid was

added and diluted to 1000ml with water.

c) Cadmium: 0.1 g of Cd metal was dissolved in 4 ml concentration nitric acid, to this 8

ml of nitric acid was added and was diluted to 1000 ml with MiliQ water.

d) Mercury: The stock solution was prepared by taking 0.1598 g mercuric chloride

(HgCl2) in water and made up to 100ml.

3.17.3. Analysis of mercury

Mercury was analysed in a mercury analyzer, Model MA-5840, Electronics

Corporation of India (ECI) Ltd, using cold vapour technique. The principle of the method

involves the reduction of inorganic mercury (Hg2+) to metallic mercury (Hg°) by SnCl2 and

HCl. The metallic mercury is driven to the absorption cell in cold. The Hg vapour will

absorb radiation, which is proportional to the concentration of Hg present in the samples.

A reagent blank prepared similarly was run and blank value noted. From the series of

standards, a graph was plotted with concentration against absorbance. Samples were

also treated similarly and absorbance noted. And from the graph concentration of

samples was found out.

3.17.4. Analysis of other metals Determinations of other metals were done using flame AAS (Atomic absorption

spectrophotometer, Varian spectra-220 Model). The wavelength was optimized for each

metal using respective hollow metal cathode lamp. The standard calibration graph was

prepared using metals standards. Air acetylene flame was used in all cases.

3.18. Microbiological characteristics;

3.18.1. Total Plate Count (TPC)

TPC of the raw material, prepared and frozen products during storage were done

as per the methods described in A.P.H.A (Speck, 1976). Appropriate dilutions of

homogenate were made in physiological saline (0.85%) and plated in triplicates on plate

count agar by spread plate technique. The inoculated plates were incubated at 37 °C for

24-48 hours. Colonies formed were counted and expressed as colony forming unit

(cfu/g) per gram of meat.

3.18.1.1. Psychrophiles (Psy)

Psychrophiles of the ice stored octopus were done as per the methods described

in A.P.H.A (Speck, 1976). Appropriate dilutions of homogenate were made in

physiological saline (0.85%) and plated in triplicates on Tryptone Soya Agar by spread

plate technique. The inoculated plates were incubated at 5 – 7 °C (refrigerated

temperature) for 5 – 7 days. Colonies formed were counted and expressed as colony

forming unit (cfu/g) per gram of meat.

3.18.2. Protocol for the isolation of Salmonella spp. from seafoods (FDA, 1992) a. Preparation of foods for isolation of Salmonella spp.

The following methods are based on the analysis of a 25 g analytical unit at a 1:9

sample / broth ratio. Aseptically weigh 25 g of sample into a 500 ml sterile flask and add

225 ml sterile lactose broth. Plug the flask and let stand 60 min. at room temperature.

Mix well by swirling and determine pH with test paper. Adjust pH, if necessary, to 6.8 ±

0.2 with sterile 1 N NaOH and incubate 24 ± 2 hours at 35 °C.

b. Isolation of Salmonella spp. Gently shake incubated sample. For raw flesh foods and highly contaminated

foods transfer 0.1 ml mixtures to 10 ml Rappaport-Vassiliadis (RV) medium and another

1 ml mixture to 10 ml tetrathionate (TT) broth. For other foods transfer 1 ml mixture to 10

ml selenite cystine (SC) broth.

c. Incubate selective enrichment media as follows Raw flesh food and highly contaminated foods incubate in RV medium for 24 ± 2

hours at 42 ± 0.2 °C and in TT broth 24 ± 2 hours at 43 ± 0.2 °C. For other foods,

incubate in SC and TT broths at 24 ± 2 hours at 35 °C. Mix and streak 3 mm loopful from

incubated TT and SC broth and RV medium on bismuth sulfite (BS) agar, Xylose lysine

desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. Prepare BS plates one day

before streaking and store in dark at room temperature until streaked. Incubate plates

for 24 ± 2 hours at 35 °C. Look for presence of colonies that may be Salmonella.

d. Typical Salmonella colony morphology Typical Salmonella colonies are as follows:

i) Hektoen enteric (HE) agar: Blue-green to blue colonies with or without black centers.

Many cultures of Salmonella may produce colonies with large, glossy black centers or

may appear as almost completely black colonies.

ii) Xylose lysine desoxycholate (XLD) agar: Pink colonies with or without black

centers. Many cultures of Salmonella may produce colonies with large, glossy black

centers or may appear as almost completely black colonies.

iii) Bismuth sulfite (BS) agar: Brown, gray, or black colonies; sometimes they have a

metallic sheen. Surrounding medium is usually brown at first, but may turn black in time

with increased incubation, producing the so-called halo effect.

If typical colonies are present on the BS agar after 24 ± 2 hours incubation then

pick 2 or more colonies. Irrespective of whether or not BS agar plates are picket at 24 ± 2

hours, reincubate BS agar plates an additional 24 ± 2 hours. After 48 ± 2 hours

incubation, pick 2 or more typical colonies, if present, from the BS agar plates only if

colonies picked from the BS agar plates incubated for 24 ± 2 hours give atypical

reactions in triple sugar iron agar (TSI) and lysine iron agar (LIA) that result in culture

being discarded as not being Salmonella (Table 3).

e. Atypical Salmonella Colony Morphology

In the absence of typical or suspicious Salmonella colonies, search for atypical

Salmonella colonies as follows:

i) HE and XLD agars: Atypically a few Salmonella cultures produce yellow colonies with

or without black centers on HE and XLD agars. In the absence of typical Salmonella

colonies on HE or XLD agars after 24 ± 2 hours incubation, then pick 2 or more atypical

Salmonella colonies.

ii) BS agar: Atypically some strains produce green colonies with little or no darkening of

the surrounding medium. If typical or suspicious colonies are not present on BS agar

after 24 ± 2 h, then do not pick any colonies but reincubate an additional 24 ± 2 hours. If

typical or suspicious colonies are not present after 48 ± 2 hours incubation, then pick 2 or

more atypical colonies (Table 4).

3.18.3. Pre-enrichment broths, culture media, reagents etc.; Lactose broth The medium was sterilized by autoclaving at 121 °C for 15'

Selective enrichment broths; Fluid selenite cystine medium (Hi Media, Mumbai) The medium was prepared as per the manufacturer's instructions.

Tetrathionate enrichment broth (Hi Media, Mumbai)

The medium was prepared as per the manufacturer's instructions.

Rappaport-Vassiliadis medium (Hi Media, Mumbai) The medium was prepared as per the manufacturer's instructions.

Selective plating media;

Bismuth sulphite agar (Hi Media, Mumbai) The medium was prepared as per the manufacturer's instructions

Hektoen enteric agar (Hi Media, Mumbai) The medium was prepared as per the manufacturer's instructions

Physiological saline Sodium chloride (8.5 g) was dissolved in 1000 ml distilled water, this was

sterilized by autoclaving at 121 °C for 15'. Trypticase soya agar (TSA), (Hi Media)

The medium was sterilized by autoclaving at 121 °C for 15'.

Triple sugar iron agar (TSI), (Hi Media) The medium was dissolved by boiling, distributed to tubes, and autoclaved at 110

°C for 15’.

Urea broth base (Hi media) This medium was dissolved, distributed to tubes, and autoclaved at 121 °C for

15'. At around 50 °C after sterilization, 1.5-3.0 ml of sterile 40% urea solution was added

to 13X100 sterile test tubes aseptically and mixed well.

Tryptone broth The medium was sterilized by autoclaving at 121 °C for 15'.

Kovac's reagent (p-DMAB) Lysine iron agar (LIA), (Hi Media)

The medium was dissolved by boiling, distributed to tubes, and autoclaved at 110 °C for

15'. The medium was then allowed to set in a sloped condition with a butt of 1".

Methyl red-Voges Proskauer broth (MR-VP), (Hi Media) The medium was dispensed in 5ml proportions into tubes and autoclaved at 121 °C for

15'.

Methyl red reagent (Hi Media) Voges Proskaur's reagent (Hi Media) Solution A and Solution B were used

Simmons citrate agar (Hi Media) The medium was dissolved by boiling, dispensed into tubes, and sterilized by

autoclaving at 121 °C for 15'. After cooling to 50-55 °C, allowed solidifying in slanted

position.

Phenol red Dulcitol broth (Hi Media)

The medium was dissolved and dispensed into tubes containing inverted

fermentation tubes and autoclaved at 110 °C for 10'.

Maintenance media

The medium was sterilized by autoclaving at 121 °C for 15'.

Liquid paraffin Sterilized in hot air oven at 180 °C for 2 hours

3.18.4. Procedure for enrichment and isolation of V. cholerae (FDA, 1992).

a) Sample preparation Blend 25 g of the sample in 500 ml with 225 ml alkaline peptone water (APW) for

2 min. Leave blended solution of sample including frozen or otherwise processed food

homogenate and dilutions, in jar or pour into loosely stoppered sterile 500 ml.

Erlenmeyer flasks and incubate for 6-8 hrs. at 35-37 °C. Reincubate enrichment broths

for total incubation time of 18-24 hrs. Plate the 18-24 hr. enrichment broth to isolate V.

parahaemolyticus.

b) Colony isolation

After incubation, without shaking, transfer 3-5 mm inoculum from surface pellicle

on the selective plate TCBS agar; incubate it for 18-24 hrs at 35-37 °C. Select 3 or more

yellow colonies such are large, smooth yellow and slightly flattened colonies with opaque

centers and translucent peripheries. Streak each isolated colony on TSA (2 % NaCl one)

and incubate for 18-24 hrs at 35-37 °C. Streaking for isolation on non-selective medium

may be necessary to ensure colony purity before biochemical testing.

c) Colony Morphology On TCBS agar V. cholerae (El Tor and classical) are large smooth yellow

(sucrose +ve) and slightly flattened colonies with opaque centers with translucent

peripheries.

Note : Vibrio spp. does not produce tiny creamy yellow colonies on TCBS agar.

d) Distinguishing suspect Vibrios from non Vibrios by biochemical tests;

i. Cytochrome oxidase test

18 - 24 hrs. old cultures from TSA slants are used for cytochrome oxidase test.

Turning of the oxidase paper into purple colour on touching young bacterial culture

indicate a positive reaction ((Table 5).

ii. H2S production in TSI, KIA slants Individual colonies are inoculated into the media by stabbing the butt and

streaking slant of TSI and KIA slants. Turning of the media into black colour indicates H2S production. This is to differentiate Vibrios from other bacteria like Aeromonas spp, Psuedomonas which do not produce H2S. iii. Oxidation-Fermentation test

Inoculate two tubes of Hugh-Leifson medium (semi solid) with growth from an isolated colony. Overlay medium in one tube with sterile mineral oil or liquid vaspar and incubate for 1-2 days. Acid causes dye to change from purple to yellow. Vibrio sp. ferment glucose oxidatively. 3.18.5. Procedure for enrichment and isolation of Vibrio parahaemolyticus (FDA, 1992)

The sample preparation for V. parahaemolyticus is typically same as V.cholerae except for incubation time and maintenance of 2-3% salt in all the media enrichment broths and diluent. Incubate for 12-16 hrs. at 35-37 °C, Streak 1 loop full from top 1 cm of enrichment broth and incubate at 35-37 °C for 18-24 hrs. Round 2-3 cm diameter green or blue green colonies could be V. parahaemolyticus. 3.19. Organoleptic evaluation The fresh, ice stored and prepared products were assessed for qualities using 9 point hedonic scale with the help of experienced panelists (familiar with such products) and regular fish eaters after production and during storage at monthly intervals. The frozen octopus meat was steam cooked for 5 min. and served to the panelist. The smoked octopus meat was warmed up and presented. Battered and breaded octopus was deep fried in refined vegetable oil and served for panelists for sensory evaluation. The opinion of the panelist was considered as the main criteria for judging the quality and shelf life of the product. The panelists were requested to give scores based on 9 point hedonic scale for appearance, colour, odour, taste, texture and overall acceptability. 3.19.1. Organoleptic scores

9 Like extremely 4 Dislike slightly 8 Like very much 3 Dislike moderately 7 Like moderately 2 Dislike very much 6 Like slightly 1 Dislike extremely 5 Neither like nor dislike

3.20. Statistical analyses Appropriate statistical analysis was done. Analyses of variance technique is used

wherever necessary using “Dry soft” software (Ramamohan, 1993) to find out the significance between the products / treatments/ methods. Pearson correlation coefficients were estimated wherever found necessary using the improved version of SPSS Version - 10 package (Panse and Sukthatme, 1967) to assess the relation and significance.