Gross chemical composition
The proportion composition of the four primary components of fish-water, protein, fat, and ash (mostly minerals)—is known as the gross chemical composition. Depending on factors including age, sex, environment, and season, fishes’ chemical makeup varies greatly both between species and within individuals within the same species. There is little difference in the amount of protein and ash. Water content has an inverse connection with lipid content, which varies remarkably.
The standard protocols for the analysis of gross chemical compositions are given below:
1.Moisture
Principle: The water content of finely minced meat or ground fish is determined by the loss in weight after heating the sample at a controlled temperature (105°C). The decrease in weight corresponds to the amount of water that evaporated from the sample. The result is expressed as grams of water per 100 grams of meat (g/100g).
Procedure
- Preparation of petridish:
- A clean, dry petri dish is pre-weighed (record this as W1) after being dried in an oven at 105°C for 2 hours, then cooled in a desiccator.
- Sample Weighing:
- Weigh 10–20g of minced meat into the pre-weighed petri dish. Record the total weight (petri dish + sample) as W2.
- Drying:
- Place the sample in an oven at 105°C overnight to evaporate moisture.
- Cool the dish in a desiccator and weigh again. Record this as W3.
- Repeat Heating:
- Return the sample to the oven for 30 more minutes, cool in a desiccator, and weigh again.
- Repeat until a constant weight is achieved (no significant change between weighing).
Calculation
To calculate the moisture content:
Moisture (%) =W2−W3 ×100
W2−W1
Moisture (%) = Weight of moisture in the sample x 100
Weight of wet sample
Where:
· W1 = Weight of empty petri dish
· W2 = Weight of petri dish + fresh meat
· W3 = Weight of petri dish + dried meat
2.Crude Protein
Principle: Nitrogenous compounds in the sample are digested with concentrated sulfuric acid, converting them to ammonium sulfate. Upon distillation with excess alkali, ammonia gas is released, which is then absorbed in boric acid. The absorbed ammonia is quantified by titration using standardized sulfuric acid. The amount of ammonia corresponds to the nitrogen content, which can be used to estimate protein content.
Procedure
1. Digestion
- Weigh 1–0.2 g of the wet sample into a Kjeldahl flask.
- Add a pinch of digestion mixture (Copper sulfate: Potassium sulfate in 1:8 ratio, finely powdered).
- Add 10 mL of concentrated sulfuric acid.
- Heat the flask on a sand bath, initially slowly, then vigorously, until the solution becomes clear and colorless.
- Cool the digested mixture and dilute to 100 mL with distilled water (or as required based on expected protein content).
- Prepare a blank with distilled water (without sample) for reference.
2. Distillation
- In a receiving conical flask, place:
- 10 mL boric acid solution
- A few drops of Tashiro’s indicator (which is pink initially)
- Ensure the tip of the condenser is immersed slightly in the boric acid.
- Pipette 5 mL (or suitable volume) of the digested sample into the distillation unit.
- Add 10 mL of 40% NaOH (or sufficient quantity to make the solution alkaline—confirmed using phenolphthalein).
- Rinse with distilled water and seal the distillation unit to make it airtight.
- Steam distill for 5 minutes or until the boric acid volume doubles.
- The boric acid solution will change from pink to green (due to ammonia absorption).
- Remove the receiving flask and rinse the condenser tip into the flask with a small volume of distilled water.
3. Titration
- Titrate the green boric acid solution with N/100 sulfuric acid.
- Continue titration until the solution turns back to its original pink color (end point).
- Record the volume of sulfuric acid used.
- Repeat distillation and titration steps to obtain concordant readings (consistent values).
Calculation
- 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 titre 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
Where,
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
Principle: Fat, being soluble in organic solvents, can be extracted from moisture-free samples using solvents like petroleum ether or ethyl ether. The solvent is then evaporated, and the residual fat is weighed (gravimetric estimation). The difference in weight before and after extraction gives the fat content.
Procedure
- Sample Preparation:
- Weigh 2-5 g (W1) of a completely dried sample into a Soxhlet thimble.
- Plug the thimble with cotton to prevent loss of sample particles.
- Soxhlet Extraction:
- Place the thimble in the Soxhlet extractor.
- Add approximately 5 volumes of ether (~200 mL) into the apparatus.
- Heat the setup and allow the solvent to reflux and extract fat continuously for about 16 hours.
- Collection:
- After extraction, allow the apparatus to cool.
- Collect the ether containing dissolved fat into a pre-weighed conical flask (W2) by filtering.
- Washing:
- Rinse the flask (that held the sample) with small quantities of ether.
- Add these washings to the same conical flask (W2) to ensure complete transfer of fat.
- Evaporation & Drying:
- Evaporate the ether from the conical flask.
- Dry the flask containing the extracted fat at 80–100°C to remove any residual solvent.
- Cool the flask in a desiccator and weigh it again (W3).
Calculation
Ash content (g/100g) = W3−W1 x 100
W2-W1
Where:
- W1 = Weight of empty crucible
- W2 = Weight of crucible + sample before ashing
- W3 = Weight of crucible + ash (after final heating)
Reference
AOAC (2023). Official methods of Analysis. Association of Analytical Chemists. 22nd Edition.