Fish Microbiology and Quality Assurance

17 . Chemical methods of assessing fish quality: Determination of hypoxanthine in seafoods

The quality deterioration of fresh fish is due to activity of autolytic enzymes and associated bacterial flora which bring about chemical changes leading to spoilage. The extent of spoilage is directly related to the accumulation of chemical substances such as ammonia, histamine, hypoxanthine, total volatile bases (TVB) and trimethyl amine (TMA). The levels for each these chemical substances can be established for fishes with different spoilage stages.

Hypoxanthine (Hx) is an important freshness index, which is formed as a result of breakdown of ATP by the enzymes. A protein free extract of fish muscle made using perchloric acid is neutralized and the perchlorate is removed as its insoluble potassium salt. The hypoxanthine present in an aliquot of the neutral solution is converted to uric acid by the enzyme, xanthine oxidase and its level determined by measuring absorbance at 290 nm using spectrophotometer.

Materials required

1. 0.6M Perchloric acid: Dilute 200 ml of conc. 60-62% acid to 3L with distilled water.
2. Potassium hydroxide – phosphate buffer: Dissolve 27.2 g of KH₂PO₄ in about 250 ml water and add 170 ml of 1.0M NaOH and adjust pH to 7.6 ± 0.5. Add 557 ml of 1M KOH to the buffer solution and make up to 1 L with distilled water.
3. 0.25M Phosphate Buffer (pH 7.6): Dissolve 17.0 g of KH₂PO₄ in about 250ml distilled water. Add 107 ml of 1.0M NaOH and check the pH and adjust to pH7.6 ± 0.5, if necessary. Make the volume up to 500 ml.
4. 0.05M Phosphate buffer (pH 7.6): Dilute 0.25M phosphate buffer (reagent 3) five fold with distilled water.
5. Xanthine oxidase working solution: To be freshly prepared each day. Commercially available stock enzyme solution having an activity of 3-4 EU/ml is diluted to 50-fold to prevent denaturation with chilled 0.05 M phosphate buffer.
6. Hypoxanthine stock standard solution(100mg/ml): Dissolve 10 mg of hypoxanthine in about 70 ml of boiling water, cool and make up to 100ml and store in a refrigerator.
7. Hypoxanthine working standards: Dilute the stock with distilled water to give working standards containing 5, 10, 15, 20, 25mg hypoxanthine in 1.0 ml. The concentration can be checked by measuring the optical density of the 10mg/1.0 ml solution at 250nm. It should be 0.786. (The molar extinction coefficient of hypoxanthine molecular weight 136.1 is 10.7 x 103).
8. Tissue blender
9. Spectrophotometer (UV-Visible)
10. Water bath at 350 +/- 2⁰C
    

Procedure

1. Weigh out accurately 5.00 ± 0.05 g of fish muscle free from skin, blood and dark muscle and blend with 50 ml of chilled 0.6M perchloric acid and filter it.
2. Take 5.0 ml of the filtrate and add 5.0 ml of KOH ^– phosphate buffer.
3. Chill and filter. Keep the extract chilled, but store in a freezer if the analysis is not to be completed the same day.
4. Prepare reaction mixture: Pipette out 0.5, 1.0 and 2.0 ml of filtrate into a test tube and make up to 2.0 ml with water, if necessary. 1.0 ml of the filtrate is sufficient for most analyses. Add 2.0 ml of 0.25 M phosphate buffer and 0.5 ml of enzyme solution. Treat this as solution A.
5. Prepare extract blank: Pipette the same volume of extract as used for A into a test tube and make up to 2.0 ml as before. Add 2.0 ml of 0.25 M buffer and 0.5 ml of water. Treat this as solution B.
6. Prepare enzyme blank: For each set of determinations, prepare a blank containing 2.0 ml of buffer and 0.5 ml of enzyme solution. Treat this as solution C.
7. Prepare buffer blank: For each set of determinations, prepare a blank containing 2.0 ml of buffer and 2.5 ml of water. Treat this as solution D.
8. Incubate all the mixtures at 350C for 30 min.
9. Measure the absorbance at 290 nm in a 10mm silica cell with water in the reference cell.
10. Calculate the nil absorbance due to uric acid after correcting for the blanks as follows.

AB = (A-D) – (B-D) - (C-D) = (A-B) – (C-D)

Calculation

1. The molar extinction coefficient of uric acid at 290 nm in a 10 mm cell is 12.4x103 . One mole of uric acid is equivalent to one mole of hypoxanthine, in 4.5 ml of the reaction mixture is:

AB X 136.1 X 106 X 4.5 / 12.4 X 103 X 103 = OD X 49.4 mg
2. Assuming that fish muscle contains 80% water, all the hypoxanthine is extracted into 54 ml of 0.6M perchloric acid. This is diluted 2 fold in step 2 of the procedure.
3. The conc. Of hypoxanthine in a 5 g sample of fish is

AB X 49.4 X 54 X 2 X 100 / V X 1000 X 5 = OD X 119 / V mg /100g
Where V is the volume of extract taken to prepare the reaction.

Enzyme check
Submitting 2.0 ml aliquots of the hypoxanthine standards to analysis should check the activity of the enzyme working solution. A plot of absorbance against mg hypoxanthine in the test solution should be linear, and the line should pass through the origin. The greedier of the line, absorbance / mg hypoxanthine, should be close to 1/49.4 is 0.0202. If the conditions are not met, the enzyme is probably inactive or the wavelength setting of the spectrophotometer is incorrect. The full calibration curve should be determined for each new consignment of enzyme but to test the working solution, reagent No.5, made up on a daily basis only the most concentrated standard need be used.