Methodology

1.0 Samples preparation

Commercial fish will be weighed between 500 to 800 g and will be purchased from fresh
market in three different zones in Sabah namely Pantai Barat (Kota Kinabalu, Ranau, Tuaran),
Pedalaman (Keningau, Beaufort, Sipitang) and Kudat (Kudat, Kota Marudu, Pitas). The five
species are Caranx Ignobilis (ikan putih), Lutjanus Argentimaculatus (ikan jenahak),
Rastrelliger Scombridae (ikan kembung), Alectis Indica (ikan cermin), and Euthynnus Affinis
(ikan tongkol). Each sample will be injected with liquid nitrogen and will be deposited into
sterile plastic bag and store in ice boxes. Flesh will be separated from skin and bones without
damaging the gut by using sterile scalpel. A 400 g of samples will be packed in sterile
polystyrene boxes and stored at -20°C until further analyses.

2.0 PHYSICAL ANALYSIS

Quality assessment scheme used to identify the quality index of fish will be done according to
Larsen et al. (1992). The quality will be accessed based on general appearance of fish skin,
bloodspot on gill cover, stiffness, belly, smell, eyes clarity, eyes shape, gills colour and gill
smell.

3.0 CHEMICAL ANALYSIS

Chemical analysis of amino acid, biogenic amines, formaldehyde and ammonia will be
analyzed on fish flesh to determine the concentration in order to evaluate fish safety level.

3.1 Amino acid

3.1.1 Chemicals

Nash’s Reagent (Nash, 1953) will be used as an indicator by diluting 15 g ammonium acetate
in a 100 ml Erlenmeyer flask with an addition of 0.3 ml of acetylacetone and 0.2 ml of acetic
acid. Nash’s Reagent is light sensitive and will be kept in dark-glass reagent bottle at all time.
Trichloroacetic acid, TCA will be used to adjust the pH of fish flesh (2.32, 3, 4, 5 and 5.68)
appropriately. A 0.1 N potassium hydroxide, KOH and 0.1 N hydrochloric acid, HCl will be used
to adjust the pH of the distillate to be in range of 6.0 to 6.5. Three ranges of working standard
0-1 mg l-1, 0-5 mg l-1 and 0-30 mg l-1 will be prepared from intermediate standard solution
of 10 µg g-1 to calibrate the graph.

3.1.2 Amino Acid Analysis

Amino acid will be analyzed following methods by Noordiana et al. (2011) with details as
follow.

Samples and standard amino acids solution will be subjected to hydrolysis by mixing 0.3-0.5 g
of samples with 15 ml of 6 N HCL. The samples will be heated in oven for 24 h at 110°C. The
samples will be left to cool at room temperature and transferred into 50 ml volumetric flask.
Sample solution will be filtered through filter paper, discarding the first 2 ml initial filtrate. For
standard amino acid, 10 ml internal standard (1 ml = 2.5 mole AABA) will be added before
being transferred into 50 ml volumetric flask. Sample filtrate will be stored for four weeks at -
20°C. Analyses will be performed by Shidmadzu high performance liquid chromatography
(Shimadzu, Kyoto, Japan) consisting of a Model SPD-6A with UV detector set to 254 nm, and a
Model C-R6A chromatopac integrator. A LiChrosopher 100 RP-18 reverse phase column (5 µm,
125 x 4 mm, E. Merck, Damstadt, Germany) will be used for the analysis of amine standard
solution samples. For HPLC analysis, a small volume of the above filtrate will be filtered
through 0.2 µm aqueous syringe filter, with 25 mm diameter. A 10 µl sample filtrate will be
pipetted into tubes (measuring 6 x 50 mm). Tubes will be placed into reaction vial and
vacuumed to 100 millitorr before adding 20 µl of fresh redrying solution (consisting of
methanol: water: ethylamine at a ratio of 2:2:1 v/v) and will be then mixed thoroughly.
Samples will be vacuumed to 70 millitorr before adding fresh 20 µl of derivation reagent
(consisting of methanol: water: triethylamine: PITC at a ratio of 7:1:1:1), mixed and tubes
returned to reaction vial and valve closed. They will be left to stand for 20 mins at room
temperature and vacuumed again to 50 millitorr. For immediate analysis of sample, 100 ml
sample diluents (consisting Na2 HPO4 ) will be added and mixed. Limited volume of 20 µl will
be used as injection volume into HPLC for samples whilst injection volume for standard
solution will be 8 µl. These samples will be left to stabilize for a few weeks at temperature
between -10 to -20°C.

3.2 Biogenic amine determination

Biogenic amine determination will be carried out according to Bhowmik et al. (2017) with
details provided below.

A slightly modified method by Yen and Hsieh (1991) will be used to determine the biogenic
amines (histamine, cadavarine, and putrescine) in fish and seafood samples as described
below: Mixture of amines standard solution consisting of putrescine dihydrochloride (182.9
mg), cadaverine dihydrochloride (171.4 mg), and histamine dihydrochloride (165.7 mg), will
be dissolved in 10 ml deionised water until final concentration of each amine is 10 mg l-1
solution. The amine standard solutions will be freshly prepared every time prior to each
experiment and will be injected accordingly for samples comparison. Each stock solution of
standard amine will be freshly prepared in deionised water in duplicates for two levels to cater
for different levels of amines found in seafood in following concentration: (0, 20, 40, 60, 80,
100 µg g-1) for all cadaverine, histamine and putrescine. The amines solution will be analyzed
using HPLC and the average peak area of chromatography from two injections of each
duplicates standard will be recorded. Linear regression will be determined by the least squares
method and the correlation coefficient will be calculated. A 20 g of seafood flesh will be
transferred into a 250 ml centrifuge tube and homogenized with Polytron type homogenizer
(LH, Ltd England) with 50 ml 6% trichloroacetic acids (TCA) for 3 min. The homogenate will be
centrifuged at 11000 rpm for 10 min at 4°C to allow precipitation and will be filtered through
Whitman No.1 filter paper. The filtrate will be placed in a volumetric flask and made up to 100
ml with 6% trichloroacetic acid. Each extract (2 ml) will be derivatized with benzoyl chloride
as described below. The benzoyl derivative of amines will be prepared according to Redmond
and Tseng (1979) with some modification. A volume of 1 ml of 2 M sodium hydroxide will be
added to 50 µl of standard amine solution, followed by 10 µl benzoyl chloride. The mixture
will be mixed on a vortex mixer and left standing for 20 min. Two ml of saturated sodium
chloride will be added and followed by extraction with 4 ml diethyl ether. After centrifugation
at 10000 rpm for 10 min at 4°C and separation by will be rinsed twice with deionised water
using a separation funnel, the upper organic layer will be transferred into clean tube and
evaporated to dryness in stream of nitrogen gas. The residue will be dissolved in 500 µl and 5
µl aliquots will be injected for HPLC analysis. Each derivatives amine standard solution
consisting of histamine dihydrochloride, putrescine dihydrochloride and cadaverine
dihydrochloride will be injected and analyzed individually and finally run simultaneously to
determine their retention times. Isocratic elution system will be used for the analyses of these
amines. The mobile phase of acetonitrile-water (60:40 v/v) at flow rate of 1.1 per min resulted
in clear separations and resolution for all of the three biogenic amines tested.

3.3 Formaldehyde determination

The modified protocol to detect formaldehyde in fish by HPLC instrument based on the
method described by Claeysa et al. (2009) and validated HPLC method by Wahed et al. (2016).

3.3.1 Sample preparation

About 5 g samples will be taken; blank and spiked formalin will beadded into the samples.
Then added 5 mL acetonitrile, analytical grade and vortex. The samples got sonicated for 30
min at room temperature (25–30°C), shaken for 30 min in a shaking water bath at room
temperature at 150 rpm, centrifuged for 5 min in 6000 rpm at 22 °C, filtered through a
Whatman filter paper

(90 mm). The upper layer of the extract of approximately 5 mL will be carefully taken. 2.5 mL
working DNPH solution and vortex will be added well. The samples will be derivatised by
shaking at 150 rpm, at 40°C for 1 h in a shaking water bath and after incubation the
supernatant got filtered by a syringe micro filter (0.45um)

3.3.2 Analytical condition of HPLC

A 10 uL sample solution will be analyzed with a Luna C-18 column (25 x 4.6 mm, 5um) with a
60% methanol solution as a mobile phase and detected at the wavelength of 355 nm. The flow
rate will be 1 mL/min and the operating time will be 13 min.

3.3.3 Standard preparation from CRM (certified reference materials)

Standard will be prepared from CRM first stock solution whose concentration will be 4815 mg/
Land a second stock solution or a working stock solution of 500 mg/L will be prepared by
dissolving 2.6 mL of CRM standard solution into 25 mL H2O.

3.3.4 Qualification and quantification of formaldehyde

The sample derivatives will be analyzed in HPLC and compared to the standard formaldehyde
retention time for qualification. The peak area of the sample solution will be substituted in
the calibration equation of the standard curve to calculate the formaldehyde concentration.

3.5 pH determination

A 10 gram of fish flesh will be weighed and homogenized thoroughly with 100 ml distilled
water for 5 minutes.

4.0 MICROBIOLOGICAL ANALYSIS

Two replicated pooled samples for both types of storage will be blended using stomacher bags
for 60 s. One gram from the pool sample will be removed and diluted with 9 ml sterile peptone
water (0.1% peptone water +0.9% saline). A 0.1 ml aliquots will be spread onto different
selective agars namely plate count agar for total aerobic counts, arginine decarboxylase agar
and ornithine decarboxylase agar for putrescine producing bacteria, lysine decarboxylase agar
for cadaverine producing bacteria, and modified Niven’s media for histamine producing
bacteria. Proteolytic counts will be determined by count on the skim milk agar. All agar plates
will be incubated for 48 h at 37°C for mesophilic counts before the colony will be counted
using a colony counter and reported as colony forming units per gram (cfu g-1).