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ANALYTICAL METHODS AND PROCEDURES FOR FISH AND FISH PRODUCTS

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D -7 (Addition) Determination of sodium nitrite by colorimetric method MAKOTO YAMAGATA & LOW LAI KIM. D -8 (Supplement): Determination of sulfur dioxide in frozen shrimp ps by colorim etric and Conway's microdiffusion method MAKOTO YAMAGATA & LOW LAI KIM.

DETERMINATION OF PHYSICAL PROPERTIES OF MEAT

DETERMINATION OF MOISTURE

INTRODUCTION

After drying, transfer the bowl with the lid partially covered to the desiccator to cool (approx. 45 min.). Benefit approx. 5 g of meat sample evenly on the sample dish and cover with filter paper held in place with a Teflon ring.

DETERMINATION OF ASH

Discard the NaOH solution and wash the sand with distilled water three times or until it is free of the alkali. Pour off the HCl solution and wash the sand 3 times with distilled water or until it is acid-free.

MEASUREMENT OF FREE AND EXPRESSIBLE DRIPS

FISH PROTEIN EXTRACTIBILITY & ITS DETERMINATION

200 is the volume (ml) of 0.1 M KCl solution used for sarcoplasmic protein extraction. 50 is the total volume (ml) of the sarcoplasmic protein aliquot after addition of 10 ml of 25% TCA.

VISCOSITY OF FISH MEAT SOL

SAMPLING AND SAMPLE PREPARATION

Read the viscometer when the pointer has stabilized and note the temperature of the meat sole. Multiply the viscometer reading by 5 if the large spindle is used or by 20 if the smaller spindle is used and express the viscosity of the meat sole in centipoise.

QUALITY ASSESSMENT OF FISH JELLY PRODUCTS AND RAW MATERIAL USED FOR PRODUCTION OF

FISH JELLY PRODUCTS

GEL STRENGTH MEASUREMENT BY FUDOH RHEOMETER

Place a test piece on the sample holder and switch on the Fudoh rheometer and the chart recorder at the same time. When the test piece is broken as indicated in the recorder chart, turn OFF the chart recorder and rheometer.

ORGANOLEPTIC ASSESSMENT BY FOLDING AND TEETH-CUTTING TESTS

Each is then folded in half and if there are no tears or breaks, further folded into four. The classification gives a subjective assessment of the resistance experienced by a trained panel when the test is bitten between the upper and lower front teeth.

WATER ACTIVITY (AW)

BY CONWAY’S MICRODIFFUSION METHOD

The choice of salts used should be those with water activity in the same range as the expected water activity of the sample. A graph of percent weight change (vertical axis) against the water activities of standard saturated salts (horizontal axis) is plotted and the water activity of the unknown sample is the intersection of the graph against the X-axis as in Fig.

TABLE  1.  Water  activity  (Aw)  of  saturated  salts  at  25°C.
TABLE 1. Water activity (Aw) of saturated salts at 25°C.

DETERMINATION OF CHEMICAL PROPERTIES OF MEAT

PROTEIN DETERMINATION BY KJELDAHL METHOD

Weigh the homogenous fish sample (1g) accurately or pipette a suitable amount of protein fraction solution (20 ml myofibrillar or sarcoplasmic protein fraction or 40 ml non-proteinaceous nitrogen fraction) and place in digestion tube. Calculate the protein nitrogen (mgN/100 g or 100 ml samples) as follows: . a) solid/semi-solid fish sample.

PROTEIN DETERMINATION BY BIURET METHOD (MODIFIED BY UMEMOTO)

PREPARATION OF CALIBRATION CURVE

It is better to use myofibrillar protein extract obtained from the same group of fish to prepare the calibration curve. The concentration of the diluted fish protein extract should be between 0.1 to 0.5 mgN/ml.

DETERMINATION OF PROTEIN CONCENTRATION OF UNKNOWN SAMPLE (FISH M YOFIBRILLAR PROTEIN EXTRACT)

Plot the absorbance values ​​of the protein solutions against the concentrations of the protein solutions to obtain the calibration curve.

DETERMINATION OF TRIMETHYLAMINE OXIDE (TMAO-N), TRIMETHYLAMINE (TMA-N),

TOTAL VOLATILE BASIC NITROGEN (VB-N) BY CONWAY’S MICRODIFFUSION METHOD

Determination of TMA-N

  • Apply sealing agent to Conway’s unit
  • Pipette 1 ml of inner ring solution into inner ring
  • Pipette 1 ml of sample extract into outer ring
  • Pipette 1 ml of neutralized 10% formaldehyde into outer ring and gently mix the outer ring solutions
  • Slant the Conway’s unit with cover
  • Pipette 1 ml of saturated K2CO3 solution into outer ring
  • Immediately close the unit and tighten with a clip
  • Mix the outer ring solutions gently
  • Stand for 60 min at 37°C in incubator
  • Titrate inner ring solution against 0.02N HCI using a micro-burette until green colour turns pink
  • Do blank test using 1 ml of 4% TCA instead of sample solution

Determination of TMAO-N

  • Take 2 ml of the sample solution into a test tube
  • Stand in a 80°C water bath for 90 sec. The violet colour should disappear
  • Add saturated KNO3 dropwise in cases where violet colour persists until it dis
  • Cool in running water
  • Transfer the solution to a 10 ml volumetric flask
  • Make up to 10 ml with washings and distilled water
  • Proceed as for TMA-N determination

DETERMINATION OF DMA-N

BY DYER’S COLORIMETRIC METHOD USING COPPER DITHIOCARBAMATE

  • SAMPLE PREPARATION
  • DETERMINATION OF DMA
  • PREPARATION OF CALIBRATION CURVE
  • CALCULATION OF DMA-N CONTENT OF SAMPLES

All reagents must be GR grade. a) 5% (v/v) carbon disulfide-toluene solution (CS2) Mix 5 ml of carbon disulfide with 95 ml of toluene. Obtain the amount of DMA-N (A ug) contained in 5 ml of sample solution from the calibration curve.

DETERMINATION OF FORMALDEHYDE IN FISH MEAT USING NASH’S REAGENT

DETERMINATION OF FORMALDEHYDE

Measure the absorbance of the solution against the blank solution at 412 nm (Blank solution contains distilled water instead of the neutralized filtrate).

PREPARATION OF CALIBRATION CURVE

DETERMINATION OF K-VALUE

Ion Exchange Chromatography Method)

  • PREPARATION OF SAMPLE
  • FOR SAMPLE PREPARATION
  • FOR ION-EXCHANGE CHROMATOGRAPHY
  • PREPARATION OF ION-EXCHANGE RESIN (SCHEME 2) a) Acetone
  • PREPARATION OF SAMPLE EXTRACT See Scheme 1
  • PREPARATION OF ION-EXCHANGE RESIN See Scheme 2
    • After all the solution A has passed into the resin, run 45 ml of solution B into the column to elute ATP, ADP, AMP and IMP
    • Collect the eluate in another 50 ml volumetric flask
    • Make up the eluates to 50 ml with solutions A and B, respectively
    • Measure the absorbance of the two eluates at 250 nm

In system 1 (Fig 1), attach a siphon tube to a column placed in a beaker containing 20 ml of distilled water. In system 2 (Fig 2), attach a separatory funnel instead of a siphon to the column and pour 20 ml of distilled water into the separatory funnel. After all of the solution A has entered the resin, run 45 ml of solution B through the column to elute ATP, ADP, AMP and IMP.

V CALCULATION

SCHEME 1. PREPARATION OF FISH MUSCLE EXTRACT

SCHEME 2. PREPARATION OF ION-EXCHANGE RESIN

SCHEME 3. SIMPLIFIED FRACTIONATION METHOD FOR K-VALUE

FRESHNESS TESTING PAPER

A portion of the meat is used in the FTP, while the corresponding portions are subjected to conventional K-value analysis. Care should be taken to standardize the reading time of the colored strips of the FTP. All materials supplied with the FTP kit are easily degraded and should be stored frozen (preferably -60°C) and in the dark.

DETERMINATION OF VOLATILE BASIC NITROGEN (VB-N), TRIMETHYLAMINE OXIDE NITROGEN (TMAO-N) AND

TRIMETHYLAMINE-NITROGEN (TMA-N) BY CONWAY’S MICRO-DIFFUSION METHOD

1 Distilled water should be boiled and cooled overnight with the mouth of the flask attached to a soda-lime trap to remove carbon dioxide (CO2). Mix 200 ml of 0.1% methyl red alcohol with 50 ml of 0.1% methylene blue alcohol and store in a brown colored reagent bottle. 2 If the stock of Ti CI3 has been stored for some time, a recovery test should be carried out with different concentrations (%) of Ti CI3 and the appropriate percentage should be used to give almost 100% recovery.

Fig.  1  Analytical  procedure  for  VB-N,  TMAO-N,  TMA-N  analysis
Fig. 1 Analytical procedure for VB-N, TMAO-N, TMA-N analysis

DETERMINATION OF AMMONIA (Colorimetric method)

With series of samples or standards, complete reagent additions in sequence on each separator before proceeding to the next. Drain the aqueous layer and pass the n-butanol layer through approx. 30 g anhydrous Na2SO4 glass funnel plugged with glass wool in 100 ml Erlenmeyer flask. 13. Measure absorbance of solution at maximum wavelength approx. 680 nm in 1 cm cell against blank as reference. If the absorbance is higher than that of the highest ammonia standard, dilute the reserved filtrate quantitatively with 2.5% phosphotungstic acid solution so that 2.0 ml of diluted solution gives absorbance below this level.

QUALITATIVE TEST OF FORMALDEHYDE (Chromotropic Acid Test)

The presence of formaldehyde is indicated by the appearance of light in deep violet (the depth of the color depends on the amount of formaldehyde present).

Fig.  1.  Distillation  unit  for  formaldehyde  determination  (chromotropic  acid  test)
Fig. 1. Distillation unit for formaldehyde determination (chromotropic acid test)

DETERMINATION OF FORMALDEHYDE BY

Accurately weigh approximately 1 g of formalin (35%, stock solution) into weighing bottle with 5 ml of distilled water and make up to 100 ml with distilled water in a volumetric flask. Standard Methods of Analysis for Hygienic Chemists - with Commentary - Authorized by the Pharmaceutical Society of Japan, Kimbara Publishing Co., Ltd.

Fig.  1.  Steam  distillation  apparatus
Fig. 1. Steam distillation apparatus

DETERMINATION OF K-VALUE BY THE FRESHNESS METER

  • Sample extraction reagents
  • Reaction reagents
  • STEP 1
  • STEP 2
  • STEP 3
  • STEP 4

34;POSITION" turns on the chart recorder to set the pin position to zero on the chart paper.

Fig.  1  Calculation  of  K-Value
Fig. 1 Calculation of K-Value

FRACTIONATION AND DETERMINATION OF FISH PROTEINS

  • Total nitrogen (tN)
  • Salt soluble protein nitrogen (sspN)
  • Meat weight used for total sarcoplasmic protein nitrogen (tspN)
  • Meat weight for residual intercellular protein & denatured protein nitrogen (rpN)
  • Actual sarcoplasmic protein nitrogen, aspN

200 is the volume (ml) of a 0.1 M KCl solution used for the extraction of sarcoplasmic protein. 2) Meat weight for non-protein compounds nitrogen (npN). 20 is the volume of the salt-soluble protein sample used for digestion. 200 is the volume (ml) of a 0.6 M K Cl phosphate buffered solution. used for the sspN extraction. True sarcoplasmic protein nitrogen, aspN. The difference between total sarcoplasmic protein nitrogen and non-protein nitrogen compounds:

Fig.  1  Extraction  and  fractionation  of  protein  in  fish  muscle
Fig. 1 Extraction and fractionation of protein in fish muscle

ANALYSIS OF OILS

SIGNIFICANCE OF ANALYSIS OF LIPIDS

EXTRACTION OF LIPIDS (MODIFIED FOLCH’S METHOD)

Add C-M mixture volume of about 3.5 times the weight of sample, and 2-3 drops of antioxidant solution. Pour distilled water, volume about a quarter of that of the extract, into the separating flask.

DETERMINATION OF TOTAL LIPID CONTENT

DETERMINATION OF PHOSPHOLIPID CONTENT

Mix the packing material in chloroform and pour it gently into the column with the aid of a glass rod. Filter with 250 ml of chloroform and collect the neutral lipids in a pre-weighed evaporating flask (elution rate: 3 drops per second). Evaporate the solvent using a rotary evaporator, dry using a rotary vacuum pump for 5 minutes, cool in a desiccator for 30 minutes, and weigh the phospholipids.

DETERMINATION OF ACID VALUE

1% phenolphthalein or thymolphthalein in ethyl alcohol Dissolve 1 g of one or another indicator in 100 ml of ethyl alcohol.

DETERMINATION OF FREE FATTY ACID (FFA)

DETERMINATION OF SAPONIFICATION VALUE

The fish lipid is extracted with C-M mixture and the solvent is evaporated using the rotary evaporator. A = titration volume of blank (ml) B = titration volume of sample (ml) F = Factor for 0.5N HCI standard solution.

DETERMINATION OF PEROXIDE VALUE

Take approximately 0.3 g of fat sample or A ml of that ekstrak which contains approximately 0.3 g of fat in a 250 ml bottle with plug.

DETERMINATION OF THIOBARBITURIC ACID (TBA) NUMBER

Dilute to 100ml with distilled water (can be kept in fridge for longer than 1 month). Place a portion of the clear aqueous solution in another test tube and read absorbance at 532 nm. Since the amount of reagent used is only 20 ml, the results must be multiplied by 0.2 to give the absorbance of the sample in 100 ml of reagent as specified by the definition.

DETERMINATION OF METHYL ESTERS OF FATTY ACIDS BY GAS CHROMATOGRAPHIC METHOD

Known mixtures of methyl esters of fatty acids or methyl esters of oil of known composition, preferably corresponding to that of material to be analyzed. Isothermal program for DEGS column Column start temperature = 180°C Column start time = 0.0 min Column program rate = 0°C/min Column end temperature = 180°C Column end time = 100 min. Injection port temperature = 200°C FID detector temperature = 200°C. Open the carrier gas tank, set the regulator outlet to 400 pKa, open the carrier valve on the GC-9A, and adjust the carrier flow rate on the GC-9A as required.

DETERMINATION OF THE DEGREE OF LIPID OXIDATION BY GAS CHROMATOGRAPHY

By measuring the ratio of C22:6 to C16:0 in the initial and subsequent steps, we can therefore use the oxidation index as a measure of the degree of docosahexaenoic acid.

PREPARATION OF METHYL ESTERS BY BORON TRIFLUORIDE METHOD

Add sample (about 350 mg preferred for GLC) to the flask, then add 0.5 N methanolic NaOH solution and anti-bubble stone. Add BF3 solution from bulb or automatic pipette through condenser and continue boiling for 2 minutes (at 90 - 100°C). Transfer approx. 1 ml of upper n-hexane solution to test tube and add a small amount of anhydrous Na2SO4 to remove H2O.

EXTRACTION AND DETERMINATION METHOD OF LEAN FISH LIPIDS

Add 100 ml of chloroform and 200 ml of methanol and then homogenize for 2 minutes with a homogenizer. Place the residue in the cup and repeat homogenization again with 50-100 ml of chloroform. This sample is used for other tests such as determination of lipid composition, fatty acid composition, fatty indices and deteriora.

Fig.  1.  Extraction  Method  of  Lean  Fish  Lipids
Fig. 1. Extraction Method of Lean Fish Lipids

LIPID COMPOSITION ANALYSIS BY THIN LAYER CHROMATOGRAPHY

These detection reagents degrade and char the lipids so that the lipid spots turn black after heating the plates. If the lipid components separated on TLC are to be determined accurately, mild detection reagents must be used and the developed spots recovered from the plate. Iodine vapor and the fluorine substances. cence materials do not break down the lipids, so the lipid spots can be recovered and the composition or quantity determined.

Fig.  1.  Kirchner’s  applicator
Fig. 1. Kirchner’s applicator

ANALYSIS OF ADDITIVES

DETECTION OF POLYPHOSPHATES

In the same way, apply 5 µl of the standard solution to the plate at a distance of 1.5 - 2 cm, but at exactly the same distance from the lower end of the plate. Remove the plate from the oven and check that there is no pungent smell of nitric acid. Measure the migration distance from the spot position to the center of the phosphate spot (A) and also to the solvent front (B).

DETERMINATION OF MONOSODIUM L-GLUTAMATE (MSG) CONTENT IN FISH JELLY PRODUCTS

PREPARATION OF L-GLUTAMIC ACID SAMPLE SOLUTION

PREPARATION OF SUPERNATANT SAMPLE IN ENZYME SOLUTION

DETERMINATION OF SUGAR (SUCROSE) BY SOMOGYI’S METHOD

  • Dilute 10 ml 1N HCI in 100 ml volumetric flask
  • Weigh 1 g NaOH, dissolve in distilled water and make up to 250 ml in a volumetric flask
  • Weigh 1 g soluble starch and 0.1 g salicylic acid, dissolve both in 99 ml distilled water
  • Weigh 2.5 g Kl, dissolve in 97.5 ml of distilled water

Mix well by swirling and place in boiling water bath for 15 minutes with aluminum foil cap.

DETERMINATION OF STARCH

  • as an extender to increase the bulk of production 2) as a binding agent
  • Dissolve 8 g KOH in 4 ml of distilled water completely and mix with 96 ml absolute alcohol. (Potassium hydroxide must be dissolved in water first as it is insoluble in
  • Dissolve 1 g soluble starch and 0.1 g salicylic acid in 99 ml distilled water. Boil to dissolve the suspension
  • Dissolve 2.5 g Kl in distilled water and make up to 100 ml
  • Weigh 1.5 g dried KIO3 accurately and dissolve in 500 ml distilled water in volumetric flask
  • To 10 ml of KIO3 solution and 10 ml distilled water (BLANK) each add 2.5% Kl (20 ml) and 2N H2SO4 (20 ml)
  • where 0.0017835 : conversion factor of 1 ml 0.05N Na2S2O3 to KIO3 A titration volume of KIO3 solution (ml)
  • pipette 10 ml into 100 ml Erlenmeyer flask

Centrifuge at 2000 rpm/5 minutes, discard ppte supernatant. transfer the ppte to a 300 ml Erlenmeyer flask with 200 ml 2.5% HCl and boil for 2½ hours in a condenser water bath, cool. pipette 10 ml into a 100 ml Erlenmeyer flask. place in a boiling water bath with an aluminum foil cover for 25 minutes. while adding it should be quick, use a Pasteur pipette) mix well. A blank with distilled water (10 ml) should be performed at the same time as the supernatant sample.

DETERMINATION OF SALT

SEMI-QUANTITATIVE ANALYSIS OF BORIC ACID AND BORATES IN MEAT AND MEAT PRODUCTS

Wash the homogenizer container with distilled water and top up to 150 ml with hot distilled water (approx. 80 °C). For the blank test, use 10 ml of distilled water instead of the sample and repeat steps 1 to 13 to obtain a blank test solution. For a blank test, pipette 20 ml of blank test solution into a 25 ml volumetric flask and repeat steps 2 to 4.

Fig.  1  Sample  Preparation  for  NO2  analysis
Fig. 1 Sample Preparation for NO2 analysis

CONWAY’S MICRODIFFUSION METHOD

Take A ml of the sodium bisulphite solution and make up to 300 ml with the dilution solution. Wash inner ring with absorbent solution and top up to 5 ml with absorbent solution.

Pigura

TABLE  1.  Water  activity  (Aw)  of  saturated  salts  at  25°C.
TABLE  2.  Graph  of  percentage  weight  change  of  Conway  unit  against  water  activities  of  saturated  salts.
Fig.  1  Graph  Insert  Method
Fig.  1  Analytical  procedure  for  VB-N,  TMAO-N,  TMA-N  analysis
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