Proximate composition of fish is generally the percentage composition of the four basic constituents viz. water, protein, fat and ash (mainly minerals). The chemical composition of fish varies widely between species and among the individual fishes within the same species depending on age, sex, environment and season. Protein and ash content do not register much variation. Lipid content shows remarkable variation and shows an inverse relationship with water content.
The standard protocols for the analysis of proximate compositions are given below:
Principle: The difference in weight after heating the finely minced meat or a ground fish at a particular temperature for a defined duration gives the water content present in the sample. It is represented as g per 100g meat.
Procedure: Pooled minced meat was taken in a clean dry petri dish, kept in an oven at 105º C for 2 hrs, cooled in a desiccator and weighed (W1). About 10-20g portion of meat (W2) was taken in the pre-weighed petri dish, kept in an oven maintained at 105º C overnight. The petri dish was cooled in a desiccator and weighed again (W3). The petri dish was again kept in an oven for half an hour, cooled as above and weighed again to get reproducible weights.CalculationsMoisture content (%):W2-W3 / W2-W1x 100Moisture content (%):Weight of moisture in the sample / Weight of wet samplex 100
2. Crude Protein
Principle: The nitrogenous compounds in the sample are converted in ammonium sulfate by boiling with concentrated sulfuric acid. Upon distillation with excess alkali, the ammonia is liberated which is estimated by titration with standardized sulfuric acid.
Digestion: 0.1-0.2g of wet sample was weighed in to a Kjeldahl flask. A pinch of digestion mixture (copper sulphate and potassium sulphate were mixed in the ratio 1:8 and finely powdered) and 10 ml of concentrated sulfuric acid was added. It was then digested over a sand bath by heating slowly till the solution starts boiling and then vigorously until the solution becomes colorless. The sample was then cooled and made up to the desired volume (100ml) according to the protein content of the sample. A blank was kept with distilled water.
Distillation: A conical flask containing 10 ml of boric acid with few drops of Thashiro’s indicator (pink in color) was placed at the receiving end of the distillation apparatus in such a way that the tip of the condenser is slightly immersed in boric acid. 5ml or any convenient volume of the made up sample was pipette out in to the distillation apparatus. 10ml or known volume of 40% NaOH as shown excess by phenolphthlene indicator was added in to the distillation unit followed by rinsing with little distilled water. The unit was made air tight. The content was steam distilled till the boric acid solution in the flask doubles or for 5minutes. The color of the solution turns green. The flask was lowered and the condenser tip was washed with little water.
Titration: The solution in the receiving flask is green at this stage. The content was titrated against N/100 sulfuric acid until the original pink color is restored. The volume of acid used for titration was noted. The distillation and titration process was repeated to get concordant value.
1000ml 1N H2SO4 =14g N2
1ml 1 N H2SO4 = 0.014g N2
1ml 0.01 N N/100 H2SO4= 0.00014g nitrogen or (0.14/1000)
If the titer value of the sample after subtracting blank is “X”, then,Protein content (%) =X x 0.14 x V x 6.25* x 100 / 1000 x V1 x W
V – Total volume of digest
V1 – Volume of the digest for distillation
W – Weight of sample for digestion
* Nitrogen content of most fish/meat protein is 16%. Hence 1 g nitrogen equivalent of protein is 100/16 or 6.25.
3. Crude Fat (Soxhelet method)
Principle: Fat soluble in organic solvents can be extracted from moisture free samples by using solvents like petroleum ether, ethyl ether etc. The solvent is evaporated and fat is estimated gravimetrically.
Procedure: 5-10g of dried sample was weighed accurately in to a thimble and cotton plugged. The thimble was then placed in a soxhelet apparatus and 1and half volume of ether (approximately 200ml) was added and distilled for 16 hrs. The apparatus was cooled and the solvent was filtered in to a pre-weighed conical flask (W2). The flask of the apparatus was rinsed with small quantities of ether and the washings were added to the above flask. The ether was removed by evaporation and the flask with fat was dried at 80-100 ºC, cooled in a dessicator and weighed (W3)CalculationFat content (g/100g) =W3-W2 / W1x 100Moisture content (%):Weight of fat / Weight of samplex 100
W1-weight of dry matter taken for extraction;
W2- weight of conical flask and
W3- weight of flask with fat
Principle: Ash is the residue obtained after incineration of the dry material at high temperature and appears as grey-white colored powder.
Procedure: Silica crucible or platinum was heated to 600 ºC in a muffle furnace for one hour, cooled in a dessicator and weighed (W1). 2g of dried sample was weighed accurately in to a crucible and heated at low flame by keeping on a clay triangle to char the organic matter (W2). The charred material was then placed inside the previously set (600 ºC) muffle furnace and heated for 6-8 hrs which gave a white or grayish white ash. The crucible was cooled in a desiccator and weighed (W3). The crucible was heated again for further 30mins to confirm completion of ashing, cooled and weighed again.CalculationAsh content (g/100g) =W3 –W1 / W2-W1x 100
W1 – Weight of crucible,
W2 – Weight of dry matter and crucible,
W3 – Weight of crucible after ashingAsh content (g/100g) =Weight of ash / Weight of samplex 100
AOAC (2000) Association of Analytical Chemists, 17th Edition.
Conceptualized, Developed and Maintained by Dr. B. P. Mohanty and D. Karunakaran
© ICAR-Central Inland Fisheries Research Institute, Barrackpore.
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