Process Biochemistry 41 (2006) 697–700

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Extraction of muscle proteins and gelatine from cod head

Jan Arne Arnesen, Asbjørn Gildberg *
Norwegian Institute of Fisheries and Aquaculture Research, N-9291 Tromsø, Norway
Received 4 April 2005; received in revised form 4 August 2005; accepted 4 September 2005

Abstract
A simple method to extract proteins from minced cod head by mild chemical treatment has been developed. The major part of muscle proteins
was recovered by successive extraction at room temperature in dilute NaOH (pH 11) and HCl (pH 2–2.6). Gelatine was extracted at acid conditions
and elevated temperatures from residual connective tissues and bones. Almost half of the total protein (47.5%) was extracted from muscle and soft
tissues, whereas 12% were recovered as gelatine from soft connective tissues and bones. Functional properties of the various gelatine fractions were
compared with functional properties of gelatine extracted from cod skin. Gelatine extracted from soft head connective tissues had similar molecular
weight, viscosity and gel strength as gelatine from cod skin. The higher temperatures and stronger acidity necessary to extract gelatine from the
head bones, resulted in more hydrolyzed gelatine with poor gelling properties, but still viscosities were only moderately lower than the viscosity of
gelatine extracted from soft connective tissues.
# 2005 Elsevier Ltd. All rights reserved.

Keywords: Cod head; Protein recovery; Fish bone; Gelatine

1. Introduction where functional muscle and connective tissue proteins could

Since the end of previous century, the total annual world fish
catch has stabilized at about 90 million tonnes, and no further
catch growth is expected in the future [1]. Hence, optimal
utilisation of the raw material is of premium importance to
serve the increasing demand for marine oils and proteins.
Although fish fillets normally yield less than 50% of the total
fish weight, other parts like viscera, backbone, skin and head
are poorly utilized by the fish processing industry. On a world
basis as much as 25% of the total marine catches are discarded
[2]. At present the utilized by-products mainly serve as low
price ingredients in animal feed production.
In cod fisheries the head is a major by-product fraction
yielding about 20% of the fish weight [3]. In Norway it is still
common practice to cut off and throw the heads overboard at
sea due to low earnings on this raw material, but at present
considerable efforts is made to reveal new possibilities for
profitable utilisation.
Cod head is a complex raw material containing about 55%
muscle, 20% bones, 15% gills, 5% skin and about 4% eyes, and
the average protein content is about 15% [4,5]. The aim of the
present work was to develop a simple bulk processing method
be recovered in separate homogenous fractions. In a previous
work [6], only limited solubilisation of bone collagen was
measured during enzymatic solubilisation of muscle from cod
backbone, but the bone gelatine extracted after such exogenous
enzyme treatment had quite low molecular weight and no
ability to form gels. Hence, in the present work specific protein
fractions were recovered by combined chemical and physical
treatments only, without applying exogenous enzymes. The
major part of muscle proteins were extracted by mild chemical/
physical treatments at room temperatures, whereas gelatine was
extracted at elevated temperatures and acid conditions.
The results reported in the present paper were achieved after
optimisation of extraction conditions during eight introductory
experiments were the recovery of general protein and gelatine
were considered to be equally important. Protein recovery was
determined in all fractions and functional properties, like gel
strength and viscosity, were determined in the different
gelatine fractions. The functional properties were compared
with the properties of other fish gelatines, and possible
applications were evaluated with reference to the literature
[7,8].
2. Materials and methods

* Corresponding author. Tel.: +47 776 29000; fax: +47 776 29100. Atlantic cod (Gadus morhua) were caught by trawl in the Barents Sea. The
E-mail address: asbjorn.gildberg@fiskeriforskning.no (A. Gildberg). weight of the fish was 3.1–4.6 kg. The fish was stored on ice for two days before

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2005.09.001
698 J.A. Arnesen, A. Gildberg / Process Biochemistry 41 (2006) 697–700

use. Heads from five fishes were comminuted in a meat mincer (Kilia). For Fig. 1, percentage of the total protein recovered in final
extraction of proteins, samples of comminuted heads (2000 g) were used. All
fractions is given. The two initial alkaline extracts yielded three
extractions and incubations were performed in an open tank (F = 17 cm) at
room temperature with stirring (2.5 cm  7 cm steel blade, 200 rpm). Fig. 1 times as much protein as the acid extract. Altogether 47.5% of
shows a flow chart of the extraction procedure, and the experimental conditions the total protein was recovered in the three-pooled extracts. The
are given in the following: major part (73%) of this protein precipitated during neutralisa-
tion, and was easily collected by centrifugation. Due to the
1. Extraction: water (2000 ml) was added and pH adjusted to 11 with 3 M protein solubilisation pH changed during extractions. More
NaOH (62 ml). After extraction for 15 min the sample was centrifuged
protein may have been recovered if pH had been kept constant
(15 min, 4000  g) at 4 8C in a Jouan KR 4i swing out centrifuge.
2. Extraction: the sediment was suspended in water (2000 ml), and pH was by adding acid or alkali, but this would imply more salt in the
adjusted to 11 with 3 M NaOH (15 ml). The sample was extracted 60 min, protein extracts. The amount of protein in all fractions (92%)
and centrifuged as given above. does not summarize to 100% because some proteins were lost
3. Extraction: the sediment was suspended in water (2000 ml), and pH was during separation and washing of bone and soft tissue. The
adjusted to 2 with 3 M HCl (145 ml). The sample was extracted (15 min) and
muscle protein recovery was much lower (7–10%) when freeze-
centrifuged as given above.
stored cod heads were minced and subjected to extraction at
The supernatants from the three extracts were pooled and pH was adjusted to 7 similar conditions (results not shown). The main reason for this
with 3 M NaOH. After precipitation (15 min, room temperature) the sample was
separated into precipitate and soluble protein by centrifugation at 4 8C (60 min,
is probably denaturation and intermolecular cross linking of
5000  g). myofibrillar proteins during freeze-storage [14]. Freeze-storage
did not seem to influence subsequent extraction of gelatine.
The solids remaining after the third extraction; bone, skin and residual
muscle tissues were washed several times with water. Because bone settles The total protein recovery in gelatine fractions was 12%,
faster than skin and meat, it was possible to separate bones from other solids. whereas 32.7% remained in solid residual fractions. Altogether
The bone fraction was dried at room temperature before it was grinded (Bosch as much as 92.3% of the total protein was recovered in final
coffee grinder) and passed through a sieve (F = 2 mm). To obtain sufficient fractions.
amount off bone gelatine, dry bone powder (350 g) was recovered from several Gelatine extracted from the skin of cod head did not differ
cod head extraction samples. The bone powder was suspended in 0.6 M HCl
(1750 ml) for 20 h at 10 8C and washed with cold water (5000 ml) to remove much from gelatine extracted from cod skin, as revealed by size
excessible acid. Average pHs and temperatures during each extraction were as exclusion chromatography (analyses not shown), but it
follows; 5.3 and 60 8C, 4.4 and 70 8C, 3.8 and 80 8C, 3.6 and 85 8C and finally contained slightly less high molecular weight protein
3.5 and 90 8C. Each extraction was performed with gentle stirring for 30 min. (>200 kDa). Also the gel strength and viscosity were
The first extraction step was carried out with addition of 350 ml water to cod
comparable (Table 2). Apparently, the initial extraction
bone tissues (about 240 g dry matter), whereas 400 ml increasingly acidified
(HCl) solutions were added in each successive step after draining off the
treatments had no significant adverse effects on the properties
gelatine solutions recovered from the preceding extraction. The gelatine of successively extracted gelatine.
extracts were filtered, demineralized, concentrated and dried as described by
Arnesen and Gildberg [9].
In the skin and residual muscle tissue fraction pH was adjusted to 4 with 3 M
HCl before gelatine was extracted at 52 8C for 2 h. The extract was filtered as
described above.
Protein was measured as Kjeldahl nitrogen using protein factors 5.714 for
pure gelatine solutions [10] and 6.25 for the other protein fractions.
Hydroxyproline in the different fractions was determined as described by
Leach [11].
Gelatine from cod body skin was obtained as described earlier [12], and
molecular weight distribution in gelatine extracts was estimated by gel filtration
chromatography on a HPLC [12]. Viscosity and gel strength were measured
essentially as described earlier [12], but in the present work a Stable Micro
Systems TA.HDi1 texture analyser was used for gel strength measurements. To
achieve sufficient amount of gelatine for viscosity measurement, bone extracts 1
and 2 were pooled. Amino acid analysis was performed on hydrolyzed gelatine
samples essentially as described by Pedersen et al. [13].
Crude lipids were determined gravimetrically by Soxhlet extraction with
petroleum benzine.

3. Results and discussion

Extractions were carried out in pilot scale experiment with

2000 g of minced cod heads. Due to minor variations in raw
materials, it was not possible to set up a number of experiments
with identical chemical extraction conditions. Hence, it was
chosen to report the results from the experiment giving optimal
total recovery of crude protein and gelatine.
Minced cod head had the following chemical composition: Fig. 1. Flow chart of the extraction procedure. Protein yield (% of total) is given
protein, 15%; ash, 6.8%; lipids, 0.15% and water, 79.4%. In by figures in boxes below each final fraction.
 J.A. Arnesen, A. Gildberg / Process Biochemistry 41 (2006) 697–700 699

Table 1
Dry weight, content and yield of hydroxyproline in head bones, the different gelatine extracts, demineralisation solution and in washing solutions
Dry weight (g) %Hydroxyproline Hydroxyproline (g) Hydroxyproline yield
Bone 350 2.0 7.07 100
Extraction 1 4.1 6.3 0.26 3.7
Extraction 2 5.0 6.3 0.31 4.4
Extraction 3 13.9 6.1 0.85 12.0
Extraction 4 22.7 6.2 1.42 20.1
Extraction 5 15.6 6.5 1.01 14.3
Bone residue 128.4 1.6 2.04 28.9
HCl solution 61.9 0.20 0.12 1.7
Wash solution 1 33.7 0.08 0.03 0.4
Wash solution 2 8.6 0 0 0

Table 1 shows the content of hydroxyproline in bone and
gelatine extracted from bone. The yields of hydroxyproline in
the two first extractions were low (3.7 and 4.4%). In the third
and fourth extract the yield increases to 12 and 20%,
respectively. Hence, tough extraction conditions or acid pre-
treatment were necessary to obtain a high gelatine recovery
from cod head bones. The fifth extraction gave a low yield
indicating that residual collagen tissues were efficiently
protected by bone mineral structures. Introductory studies
showed that increasing the temperature alone without lowering
the pH gave little improvement in the recovery of gelatine from
fish bone. Extensive demineralising also proved to be necessary
to achieve good gelatine recovery.
All bone extracts yielded gelatine with similar hydroxypro-
line content (6.1–6.5%). According to hydroxyproline mea-
surements the total gelatine recovery in the extracts from head
bones was 54.5%, whereas 28.9% still remained in bone
residues. Only minor amounts of hydroxyproline were detected
in the HCl and washing solutions.
Bone particle size has a certain effect on gelatine extraction
yield. Nicolas-Simonnot et al. [15] reported highest recovery
when quite small particles (0.125–0.25 mm) were extracted.
The grinding equipment applied in the present work gave
variable particle size (F < 2 mm), and a lower average particle
size would probably give a higher gelatine recovery during
extraction.
Table 2 shows that the gelatine gel strength was reduced with
increasing extraction acidity and temperature. The gelatine
extracted at high temperature and low pH (extractions 3 and 4)
did not form a gel at 10 8C, and gelatine from the fifth
extraction did not form a gel even at 4 8C. Generally reducing
temperature from 10 to 4 8C gave 5–12 times higher gel
strength. Quite similar viscosities were recorded in gelatines
from the four initial extractions of bone. This is remarkable
considering the great differences in gel strength obtained with
the same gelatine samples.
Most probably the reduced gel strength obtained with
increasing extraction temperatures and acidity was due to a
higher rate of chemical protein hydrolysis. Results obtained by
size exclusion chromatography of the gelatine fractions support
this suggestion (Fig. 2). As expected, the molecular weight size
distribution indicated successively smaller peptide sizes with
increasing number of extraction step, but to improve readability
of the figure, the results from steps 2 and 4 were left out.
Muyonga et al. [16] have also reported that gelatine extracted
from bone of Nile perch are more hydrolyzed than gelatine
extracted from the skin.
Table 3 shows minor differences in the amino acid
composition between gelatines prepared from cod skin and
bone. Gelatine from bone had the same amount of imino acids
(proline and hydroxyproline) as gelatine from skin, and

Table 2
Viscosity and gel strength of the different gelatine extracts from cod head
Viscosity Gel strength Gel strength
at 60 8C (mps) (g) 10 8C (g) 4 8C
Soft connective 31.2  3.6 24.7  0.4 123.2  2.9
tissues
Bones
Extraction 1 24.0  3.7a 13.4  0.3 90.9  1.6
Extraction 2 5.2  0.1 63.2  1.4
Extraction 3 25.2  3.0 4.7  0.3 45.6  1.0
Extraction 4 24.4  3.3 NMb 28.5  0.7
Extraction 5 18.0  3.7 NM NM
a
Extractions 1 and 2 are pooled. Fig. 2. Size exclusion chromatogram of gelatine extracted from cod skin and
b
NM not measurable (no gel). from extractions 1, 3 and 5 of gelatine from head bones.
700 J.A. Arnesen, A. Gildberg / Process Biochemistry 41 (2006) 697–700

Table 3 Acknowledgement
Amino acid composition of gelatine from cod skin and bone
Amino acid Skina Bone Financial support from The Research Council of Norway is
Aspartate 5.2 5.3 acknowledged.
Glutamate 7.1 7.3
Hydroxyproline 5.6 5.6 References
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