Amino acid composition
Principle: Proteins in the sample are hydrolyzed into their constituent amino acids using 6N hydrochloric acid (HCl) under heat. The resulting amino acids are separated and quantified using High Performance Liquid Chromatography (HPLC), typically using a reverse-phase C18 column and a fluorescence detector. The amino acids are identified by comparing their retention times and peak areas with a known amino acid standard.
- Fish samples (50 mg) were hydrolyzed with 5 mL of 6N HCl under anaerobic conditions at 110 °C for 12 hours. Following hydrolysis, the samples were neutralized with 6N NaOH. The neutralized hydrolysates were then derivatized using an AccQ-Fluor Reagent kit (WAT052880, Waters, Milford, MA, USA).
- The derivatized samples were analyzed using an HPLC system (1525, Waters) equipped with a C18 reverse-phase (RP) column and a fluorescence detector (2475, Waters). A total of 17 amino acids were identified and quantified by comparing retention times and peak areas with those of a standard amino acid mixture (WAT088122, Waters). Tryptophan was not included in this analysis due to its degradation under acid hydrolysis.
Estimation of Tryptophan
Principle: Under acidic conditions, sucrose decomposes to 5-hydroxyfurfural, which forms a pale green condensation product with thioglycolic acid. This product further reacts specifically with tryptophan in the hydrolyzed protein to form a pink-colored complex. The intensity of the color, which is proportional to the tryptophan concentration, is measured spectrophotometrically at 500 nm.
Procedure
1. Sample Preparation
- Weigh 200 mg of finely homogenized fish mince into a test tube.
- Add 10 mL of 5% NaOH.
- Purge with nitrogen, seal the tube, and hydrolyze at 120 °C for 24 hours in an oven.
- After hydrolysis, neutralize the solution to pH 7.0 using 6N HCl.
- Make the total volume up to 100 mL with distilled water.
- Filter the hydrolysate using Whatman No. 1 filter paper.
2. Estimation of Tryptophan
- In a clean test tube, add:
- 4 mL of 50% sulfuric acid (H₂SO₄)
- 1 mL of 2.5% sucrose solution
- 1 mL of 0.6% thioglycolic acid
- Incubate the mixture in a water bath at 45–50 °C for 10 minutes.
- Add 1–0.8 mL of the prepared sample hydrolysate (aliquot) to the above mixture.
- Make the volume up to 5 mL using 1N HCl.
- Allow the solution to stand for 5 minutes.
- Measure the absorbance at 500 nm using a spectrophotometer.
Reference
- Ishida Y, Fujita T and Arai K (1981) New detection and separation methods for amino acid by high performance liquid chromatography. J Chromatography 204:143-148
- Sastry CSP and Tammuru MK (1985) Spectrophotometric determination of tryptophan in protein. J Food Sci Technol 22:146-147.