Fish Microbiology and Quality Assurance (2+2)

18.Chemical methods of assessing fish quality : Determination of histamine in seafoods by fluorometric method

Histamine is formed in seafoods due to the degradation of aminoacid, histamine by the action of spoilage bacteria possessing the enzyme histidine decarboxylase. Histamine poisoning is commonly associated with scombrid fishes and the level of histamine increases as the bacterial spoilage progresses. The estimation of histamine involves extraction by perchloric acid, followed by n-butanol under alkaline condition and finally collecting it in acid- aqueous phase. The histamine in the acid-aqueous phase on condensation with o-opthalaldehyde under alkaline condition emits fluorescence, which is read at an excitation wavelength of 340 nm and emission wavelength of 425 nm in a spectrofluorometer.

Materials required

1. 0. 4N Perchloric acid (HClO₄ )Solution: Dissolve 10 ml of conc. HClO₄ in 100 ml of distilled water
2. 0.1N HCl Solution : 10 ml of 1N HCl is made up to 100 ml with distilled water in a volumetric flask ( 1N HCl is prepared by taking 11 ml of conc. HCl in a volumetric flask and making up to 100 ml with distilled water)
3. 1N NaOH Solution : Dissolve 4 g of NaOH in 100 ml of distilled water
4. 0.1N NaOH Solution : Dilute 10 ml of 0.1N NaOH in 100 ml of distilled water
5. Sodium Chloride salt
6. Solvents : n-Butanol and Petroleum ether
7. 1 % ortho-Opthaldehyde solution (OPT): Dissolve 0.05 g of OPT in 5 ml of methanol
8. 0.2M Citric acid: Dissolve 4.20 g of citric acid in 100 ml of distilled water
9. Histamine standard:

Stock solution (1000 ppm): Dissolve 16.55 mg of histamine dichloride in 10 ml of 0.1N HCl solution
Working solution (10 ppm): Pipette out 1.0 ml of the stock and make up the volume to 100 ml with 0.1N HCl solution in volumetric flask

Procedure
Preparation of standard
Take 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the histamine working standard along with blank (2.0 ml of 0.1N HCL) in separate test tubes and make up the volume to 5ml with 0.1N HCl.
Preparation of sample
Homogenize 5 g of fish sample with 25 ml of 0.4 N HClO₄ in a homogeniser for 1 minute and centrifuge the homogenate for 10 min at 3000 rpm. Filter the supernatant through Whatman No. 1 filter paper under slight suction and make up to 50 ml with 0.4 N HClO₄.

Extraction of histamine from sample
Transfer 5 ml of the extract into 250 ml separating funnel, add 10 ml of distilled water and make alkaline with 5 ml of 1N NaOH. Then, add 1.5 – 2.0 g of NaCl (to saturation) and extract two times with 25 ml of n-butanol. Collect all the butanolic phases. Then, wash the butanolic phases with 10 ml of 1N NaOH and 2.0 g NaCl. Remove the aqueous phase and add 25 ml of petroleum ether and shake well. Discard the aqueous phase. Then, extract again the butanolic phases five times with 10 ml of 0.1N HCl. Collect the aqueous-acid phases (0.1N HCl) in a 50 ml volumetric flask and make up to 50 ml with 0.1N HCl.

Condensation reaction
Transfer 2.0 ml of each standard solution, sample and blank in separate test tubes, add 4.0 ml of 0.1N NaOH and shake well. Then, add 0.2 ml of OPT solution immediately. Shake to mix thoroughly and let stand for 5 min. Then, add 2.0 ml of 0.2 M Citric acid to the tubes and shake well. Finally, measure the fluorescence at the excitation wavelength of 340 nm and emission wavelength of 425 nm in a spectrofluorometer.

Calculation:

Draw the standard graph by plotting the concentration of the histamine (mg) in the X axis and the fluorescent intensity (mV) on the Y axis. Compute the concentration present in the sample extract from the standard graph and express as mg per gram of sample.