Presented in the paper is the standard procedure fish protein extractability and its determination. Specifically, the procedures in sampling and sample preparation, apparatus and reagents required, the protein extractability procedures and calculations for each method are presented

Lim, Pang Yong

Aquatic Commons

FISH PROTEIN EXTRACTIBILITY & ITS DETERMINATION
LIM P.Y.
INTRODUCTION
Fish proteins gradually become denatured in cold storage. The rate of denaturation 
depends largely upon storage temperature.
Badly frozen stored fish are easily recognizable. The appearance of the thawed product, 
instead of being glossy and translucent, is dull and opaque and the texture, no longer firm and 
elastic, becomes soft and spongy. The cooked flesh loses its succulence and becomes dry, 
fibrous and tasteless.
The main proteins of fish flesh are called myosin and actin. They are responsible for the 
mechanism of contraction and relaxation of muscles and are called myofibrillar proteins. 
Muscle also contains many other proteins, the sarcoplasmic protein which are soluble in tissue 
fluid and in any salt solution. During freezing and cold storage, the proteins are affected, 
especially the myofibrillar protein, resulted in the textural changes of flesh.
The myofibrillar protein extractibility, therefore is used as a quality index for the 
assessment of frozen fish. It is expressed as follows:-
Extractibility (%) = MPN – SPN x 100 (A)TN – NPN
where: MPN = myofibrillar protein-nitrogen (N mg/100 g sample)
SPN = sarcoplasmic protein-nitrogen (N mg/100 g sample)
TN = total nitrogen (N mg/100 g sample)
NPN = non proteinous nitrogen (N mg/100 g sample)
The protein extractibility is applicable to fish and its product in general and can be used as 
an indicator of the degree of protein denaturation for demersal and pelagic species during cold 
storage.
I  SAMPLING AND SAMPLE PREPARATION
Take a representative sample 22 g or more from the product. Place the sample in 
polyethylene bag and store in refrigerator or in ice so as to maintain sample integrity before 
preparation for analysis.
The dark meat, if any, should be removed prior to homogenisation of fish flesh.
Comminute the sample until homogeneous and place the homogenate in a polyethylene 
bag. Store the sample in a refrigerator or in ice until required. Ensure that the prepared 
sample is still homogeneous prior to weighing.
A-5.1
II APPARATUS
Chopper or mincer
Analytical balance, decimal to 0.1 mg
Spatula
Bottom-drive homogeniser (Nihon Seiki SN-03) or equivalent
Refrigerated centrifuge, capable of centrifuging at 12,500 g
Beakers, 100 and 250 ml
Bulb pipettes, 10, 20 & 40 ml
Glass funnels 0  60 mm
Whatman filter paper, No. 41
III REAGENTS
a) Phosphate buffer solution
0.03 M potassium di-hydrogen phosphate, 1 litre.
0.03 M di-sodium hydrogen phosphate, 1 litre.
Mix the above solutions into a 2 litre beaker.
Adjust the pH to 6.85 using these solutions.
Store in refrigerator.
b) 0.1 M potassium chloride solution
Weigh KCl required accurately, use distilled water as solvent.
c) 0.5M potassium chloride buffered solution
Accurately weigh KCl required. Dissolve the weighed KCI in the required phosphate buffer 
solution.
d) Trichloroacetic acid solution (25%, w/v)
Dissolve 25 g TCA in 75 ml distilled water.
IV PROCEDURE OF PROTEIN EXTRACTIBILITY
1. Total nitrogen. Accurately weigh a duplicate of 1 g the homogeneous fish sample for 
protein digestion (refer to protein determination by Kjeldahl method B-1.)
2. Sarcoplasmic protein nitrogen. Accurately weigh 10 g of the homogeneous fish sample. 
Blend the sample with 200 ml of 0.1 M KCI solution with the homogeniser for 4 mins 
(speed set at scale 2). Leave to stand in iced water for 2 hrs. Centrifuge the blended 
sample at 12,500 g or 9,000 rpm at 5°C for 20 mins. Pipette 20 ml supernatant 
(sarcoplasmic protein fraction) for digestion (refer to Kjeldahl method).
3. Myofibrillar protein nitrogen. Accurately weigh 10 g of the homogeneous fish sample. 
Blend sample with 200 ml 0.5 M KCl phosphate buffered solution with the homogeniser for 
4 mins (speed set at scale 2). Leave to stand in iced water for 2 hrs. Centrifuge the blended 
sample at 12,500 g or 9,000 rpm at 5°C for 20 mins. Pipette 20 ml supernatant (myofibrillar 
protein aliquot) for digestion (refer to Kjeldahl method).
4. Non proteinous nitrogen. Pipette 40 mi of sarcoplasmic protein aliquot (see Section IV 2) 
into 100 ml beaker. Add 10 ml 25% TCA and leave to stand in iced water for 30 mins with 
occasional swirling. Filter the content of the beaker with Whatman No. 41 ashless filter 
paper. Pipette 40 ml of filtrate for digestion (refer to Kjeldahl method).
A-5.2
V CALCULATIONS
Ws in formula (1) in the protein determination by Kjeldahl method (B-1 Section IV) has to be 
replaced by the meat weight (g) in each of the protein aliquot used as follows:-
1) For myofibrillar protein nitrogen (MPN)
WMPN = W1 x
20
W1 + 200
where W1 = weight of fish flesh (g) used for myofibrillar protein extraction
20 is the volume (ml) of sarcoplasmic protein aliquot used for digestion
200 is the volume (ml) of 0.5 M KCI — buffered solution used for the extraction 
of myofibrillar protein
2) For sarcoplasmic protein nitrogen (SPN)
WSPN = W2 x
20
W2 + 200
where W2 = weight of fish flesh (g) used for sarcoplasmic protein extraction
20 is the volume (ml) of sarcoplasmic protein aliquot used for digestion
200 is the volume (ml) of 0.1 M KCl solution used for the extraction of 
sarcoplasmic protein
3) For non-proteinous nitrogen (NPN)
WNPN = 40a x
40b
X
W2
50 200 + W2
where 40a is the volume (ml) of supernatant of sarcoplasmic protein aliquot used for 
non-proteinous nitrogen
40b is the volume (ml) of filtrate used for digestion taken from the sarcoplasmic 
protein aliquot after preciptiated by 25% TCA
50 is the total volume (ml) of sarcoplasmic protein aliquot after addition of 10 
ml 25% TCA
W2 = weight of fish flesh (g) used for sarcoplasmic protein extraction
200 is the volume (ml) of 0.1 M KCl solution used for the extraction of 
sarcoplasmic protein
Use the above equivalent meat weights and the formula (1) in protein determination by 
Kjeldahl method (B-1 Section IV) for calculation of respective protein nitrogen aliquot, express 
in mgN/100 g and calculate the extractibility by formula (A).
N.B. The pH plays an important role in the extraction of fish protein. The optimum pH is 
between 6.5-7.0. Adjustment of the pH of the buffer solution is important and can be 
achieved by using di-sodium hydrogen phosphate, potassium di-hydrogen phosphate or 
sodium carbonate etc.
A-5.3


Determination of physical properties of meat: Fish protein extractibility & its determination

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Determination of physical properties of meat: Fish protein extractibility & its determination

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