Food Chemistry 81 (2003) 257–262

www.elsevier.com/locate/foodchem

Isolation of collagen from the skins of Baltic cod (Gadus morhua)

Maria Sadowska*, Ilona Ko•odziejska, Celina Niecikowska
Department of Food Chemistry and Technology, Chemical Faculty, Gdańsk University of Technology,
G. Narutowicza 11/12, 80-952 Gdańsk, Poland

Received 10 May 2002; received in revised form 2 August 2002; accepted 2 August 2002

Abstract
The aim of the investigations was to establish the optimum conditions for the isolation of odourless and colourless collagen from
the skins of cod (Gadus morhua) by solvent extraction. It was established that, with the use of a one-stage, 24-h extraction of whole
skins with acetic or citric acid at ratios of material to solvent of 1:6 and 1:4, respectively, about 20% of collagen was extracted.
Using three consecutive 24-h extractions of whole skins with citric acid, 85% of collagen protein could be separated. The thermal
solubility of collagen depends on the medium applied. About 85 and 15% of collagen contained in the skins were dissolved in 0.45
M NaCl solution and water, respectively, after 24 h incubation at 30  C. The addition of 0.5% non-ionic detergent to all extracting
solutions used in the purification procedure and the solubilisation of collagen in 0.5 M citric acid entirely prevented development of
rancid off-odour for up to 50 days of storage of the product at 4  C. Similar effects could be achieved at 4 and 20  C, respectively, in
the presence of 0.14% of aseptine M-14 in the collagen solution in citric acid.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Baltic cod; Collagen skin; Isolation; Thermal solubility

1. Introduction of fish skins (Bailey & Light, 1989; Sikorski, Scott, &
Buisson, 1984; Yamaguchi, Lavety, & Love, 1976; Yata,
Splits of the skins of cattle and pig, as well as bones, Yoshida, Mizuta, & Yoshinaka, 2001). Therefore the
are the main sources of collagen and gelatin used in the methods used for isolation of collagen from cattle skins
food, pharmaceutical, cosmetic and leather industries are not effective enough for the isolation of collagen
(Bailey & Light, 1989; Cavallaro, Kemp, & Kraus, 1994; from fish skins.
Hood, 1987; Slade & Levine, 1987; Stainsby, 1987). The In the literature, there are only a few papers dealing
outbreak of mad cow disease has resulted in anxiety with the practical utilisation of connective tissue offal of
among users of cattle gelatin, due to the not-fully-con- sea vertebrates and invertebrates (Gómez-Guillén &
firmed hypothesis, that the infective agent can be trans- Montero, 2001; Gudmundsson & Hafsteinsson, 1997;
ferred from animals into human beings. Additionally, Ko•odziejska, Sikorski, & Niecikowska, 1999; Montero,
the collagen obtained from pig’s bones cannot be used Alvarez, Marti, & Borderias, 1995; Nagai & Suzuki,
as a component of some foods for religious reasons. 2000; Norland, 1990; Skrodzki, Kowalska-Gwardys,
Therefore there is a strong need to develop alternative Michniewicz, & Kujawa, 1989). Other papers, concern-
collagen sources. Fish offal, such as bones, skins, ing collagen of sea animals, focus mainly on cognitive
scales, fins, as well as the skins and collagenous mem- aspects, such as collagen content in tissues, its genetic
branes of squid separated during mechanical proces- types, amino-acid composition, extent of intra- and
sing, can serve as an alternative source of connective intermolecular crosslinking, susceptibility to endo- and
tissue, but they have not been rationally utilised up till exogenous enzymes, and thermal stability of collagen
now. The physical and chemical properties of collagen structure (Bracho & Haard, 1990; Love, Yamaguchi,
from cattle skins are substantially different from those Creach, & Lavety, 1976; Sato, Yoshinaka, & Sato, 1989;
Sikorski et al., 1984; Yamaguchi et al., 1976; Yata et al.,
* Corresponding author. Fax: +48-58-3472694. 2001). These factors influence the texture of the raw and
E-mail address: [email protected] (M. Sadowska). processed materials.
0308-8146/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0308-8146(02)00420-X
258 M. Sadowska et al. / Food Chemistry 81 (2003) 257–262

For physico-chemical studies and for many practical in 10% NaCl solution at room temperature for 24 h and
uses, pure, colourless and odourless preparations of the material was bleached with 1% H2O2 solution in
collagen, which are free of other components of con- 0.01 M NaOH.
nective tissue, are required. The objective of the investi-
gations was to establish the optimal conditions for the 2.4. Isolation of collagen using acetic acid
isolation of odourless and colourless collagen from the
skins of Baltic cod (Gadus morhua). The method of direct collagen extraction from fish
skins, with different concentrations of acetic acid at dif-
ferent ratios of material to solvent, was used. According
2. Materials and methods to Fig. 1 minced skins were slowly stirred with acetic
acid solution at 0  C; then, after 2 h, they were homo-
2.1. Raw material genized and stirred for 24 h. After the next homo-
genisation the material was centrifuged. In the case of
The skins of fresh Baltic cod (Gadus morhua) were the whole skins, the first homogenisation was omitted.
mechanically separated. The residue of adhering tissues Hydroxyproline was determined in the supernatants.
was removed manually. After thorough mixing of the
skins, samples (approximately 500 g) were prepared, and 2.5. Isolation of collagen using citric acid
stored at 20  C in polyethylene bags. For chemical
analysis and for the determination of protein solubility, The method of Skrodzki et al. (1989), with modifi-
the frozen samples were minced in a meat grinder, using cation, was used for extraction of collagen from the
mesh diameter f=3 mm. The dry weight, total protein skins with citric acid. The raw material, after washing
and hydroxyproline in raw material were determined. with water and disinfection with 0.3% H2O2, was
extracted with 0.5 M citric acid solution. Three extrac-
2.2. Determination of soluble non-collagenous compounds tions of the raw material, instead of one as proposed by
Skrodzki et al. (1989), were used and all the operations
Soluble non-collagenous substances were removed were performed at 4  C instead of at room temperature
from the skins as follows: minced or whole material was (Fig. 2). The sediment and the extract were weighed
mixed with water (1:6) for 3 min at 0  C. After separa- after the extractions, in order to calculate the nitrogen
tion of water, whole skins were gently stirred with 0.45 balance. Total nitrogen content, hydroxyproline and
M NaCl or 0.01 M NaOH solutions (1:6) for 3 min and dry weight were determined in all extracts and in the
solutions were separated by filtration. The minced skins residue after extraction.
were homogenized in the same solutions for 4 min at Collagen, dissolved in citric acid, was purified by dialysis
6000 rpm. The process of extraction with 0.45 M NaCl in distilled water at 4  C. The precipitated collagen fibres
or 0.01 M NaOH solutions was repeated twice. After were dehydrated with acetone. Total nitrogen and hydro-
each extraction, the samples were washed with water xyproline were determined to establish the conversion
(1:6) at 4  C. All mixtures were centrifuged for 30 min at
2000g at 0  C and subsequently, in combined super-
natants from each treatment, the hydroxyproline and
total nitrogen were determined.

2.3. Removing of lipids and pigments

To obtain odourless collagen from the cod skins,

lipids were removed by adding 0.5% of detergents to all
solutions used for the treatment. The following method
described by Gudmundsson and Hafsteinsson (1997) was
also used; the skins were treated three times with each of
the following solutions: 0.2% NaOH, 0.2% H2SO4 and
0.7% citric acid (1:7). After each extraction the skins
were washed with water until pH 7 was reached.
The method of Montero et al. (1995) for removal of
pigments from plaice skins was applied; it is based on
repeated homogenization of skins with 0.4 M NaCl
solution. Also, the usefulness of a procedure elaborated
by Ko•odziejska et al. (1999) for collagen isolation from Fig. 1. Flow sheet of the procedure used for isolation of collagen from
squid skins was studied. Skins of Baltic cod were soaked minced, homogenised cod skins with acetic acid. 1Skins: solutions.
 M. Sadowska et al. / Food Chemistry 81 (2003) 257–262 259

3. Results and discussion

3.1. Chemical composition of cod skins

The gross composition of skins of cod caught in the

same season in different years is almost constant
(Table 1). The established conversion factor for calcu-
lating the content of cod skin collagen from hydro-
xyproline was 14.7. The collagen content in the skins
amounts, on average, to 21.5% in the wet material and
71.2% in the dry weight. The established conversion
factor of nitrogen to collagen was 6.25, the same as used
for calculating of non-collagenous proteins. Considering
the share of collagen in total protein, determined on the
basis of hydroxyproline in samples, it can be assessed
that the skins, on a wet and dry weight basis contain,
respectively, 4.9 and 16.3% of non-collagen proteins,
peptides and free amino-acids. These nitrogen com-
pounds can be separated entirely by extracting minced
skins with 0.01 M NaOH and water (Table 2). 0.45 M
Fig. 2. Flow sheet of the procedure used for isolation of collagen from
NaCl solution is a less effective solvent for albumins and
whole cod skins with citric acid. 1Solutions:skins; 2dry weight.
globulins in frozen skins, probably due to denaturation
of these proteins in the course of extractions.
Both the collagen and non-collagenous protein con-
factor of nitrogen and hydroxyproline to collagen in the tents in cod skins depend upon the fishing season. Dur-
skins. ing starvation, albumins and globulins are degraded and
the collagen content in skins increases (Love et al., 1976;
2.6. Thermal solubility of collagen Sikorski et al., 1984). This fact could be responsible for
the discrepancy between our results and the results of
Thermal solubility of collagen from skins, in the tem- Young and Lorimer (1960), who found the collagen and
perature range 0–40  C, was studied. The samples, non-collagenous protein contents of cod (Gadus call-
minced as described above, were suspended in water or arias) skins to be, respectively, 74.4 and 12.9% on a dry
0.45 M NaCl solution (1:6) and incubated for 24 h in a
water bath at a given temperature with intermittent
Table 1
mixing. Subsequently, the samples were centrifuged at Proximate composition of skins of Baltic coda
10,000g for 30 min at 15  C. Hydroxyproline in the
supernatants was determined. Skins of Baltic cod Dry weight Nitrogen Hydroxyproline
caught in autumn [%] [%] [%]

2.7. Dry weight and total nitrogen 1997 30.11.0 4.200.1 1.490.01
1999 30.31.3 4.340.2 1.390.05
The dry weight and total nitrogen were determined a
Mean valuestandard deviation from three separate samples.
according to AOAC methods (1990).

2.8. Protein contents in extracts Table 2

Protein and hydroxyproline extracted from skins with 0.45 M NaCl or
The protein content in extracting solutions (0.45 M 0.01 M NaOH at 0  Ca
NaCl and 0.01 M NaOH) was determined according to Skins Protein Hydroxyproline Protein Hydroxyproline
the method of Lowry, Rosebrough, Farr, and Randall
extracted from 100 g of skins with
(1951).
0.45 M NaCl+water 0.01 M NaOH+water
2.9. Hydroxyproline
[g] [mg] [g] [mg]

Hydroxyproline was determined after hydrolysis of Whole 1.90.08 7.00.56 4.20.23 8.33.41
the material in 6 M HCl for 6 h at 105  C, using the Minced and 3.10.17 34.81.74 4.90.19 76.53.82
homogenised
colorimetric method recommended by ISO (Anony-
mous, 1978). a
Mean valuestandard deviation from three separate samples.
260 M. Sadowska et al. / Food Chemistry 81 (2003) 257–262

weight basis. The rest of the dry weight consisted of ash, total content of collagen, about 90%, can be extracted
12%, lipids, 1% and mucopolysaccharides, 0.5%. by treating minced, homogenized skins with 0.5 M ace-
tic acid, at 4  C for 24 h, using a ratio of skins to solvent
3.2. Isolation of collagen from Baltic cod skins of 1:40; with the ratio of 1:6 only 60% of collagen was
extracted. Using the same conditions for the whole
One of the most important features of collagen, with skins, only 40 and 20% of collagen, respectively, were
regard to its use, is colour. Collagen entirely devoid of extracted. Mincing and homogenisation of skins had a
colour is difficult to obtain because of the presence of crucial influence on the yield of collagen extracted.
pigments in fish skins. Application of the method Because of collagen characteristics, fish skins are diffi-
described by Montero et al. (1995) for removing pig- cult to mince in a meat grinder. In order to avoid ther-
ments from plaice skins was only partially successful for mal denaturation of collagen, the skins should be
processing Baltic cod skins, since the pigments were ground frozen. However, cod skin could be comminuted
only partially removed. Similarly, the procedure of col- easily after previous treatment with 0.25 M acetic acid
lagen extraction from squid skins described by Ko•od- (1:6) at a temperature below 15  C for 2 h. The loss in
ziejska et al. (1999) was not effective for treatment of yield of collagen extraction, after such preliminary
cod skins. treatment, was only about 1.5%.
The most effective way to separate the pigments from The extractability of collagen in citric acid, after a 24
collagen is to extract collagen directly from cod skins h treatment, did not depend on the ratio of skins to
with organic acids. Leaching of cod skins with 0.5 M solvent (Table 4) in the range studied of 1:4–1:20 and
acetic acid at 4  C for 24 h, followed by homogenisation was about 25%. With the use of three 24-h extractions
and centrifuging, leads to a colourless collagen solution, of the same skins with citric acid, about 85% of collagen
while the pigments remain in the precipitate. was dissolved (Table 4). The extractability of collagen
The extractability of collagen depends both on the from cod skins in citric buffer, pH 3.5, determined by
concentration of acetic acid (Table 3) and on the ratio Yamaguchi et al. (1976) after extensive homogenisation
of skins to acid (Fig. 3). The largest percentage of the
Table 4
Table 3
Solubility of nitrogen compounds of skins of Baltic cod in citric acid
Effect of acetic acid concentration on extractability of collagen from
solution
minced skins of Baltic coda
Skins:solution Number of Total nitrogen Hydroxyproline Dry weight
Concentration of pH Collagen extractability
extraction [%] [%] [%]
acetic acid [M] [% of the content in
the raw material]b 1:4 I 24.4 21.4 24.9
II 48.2 41.4 43.0
0.10 4.2 521
III 21.1 14.2 17.9
0.25 3.8 541
Residue 15.3 16.4 16.5
0.50 3.5 591 P
109 93.4 102.3
a
Minced material was homogenized after 24 h treatment with ace-
tic acid (1:6) at 4  C. 1:7 I 19.3 11.3 12.9
b
Mean valuestandard deviation from three separate samples. II 51.0 39.3 45.5
III 17.6 20.9 17.1
Residue 21.4 24.6 24.8
P
107.3 96.1 100.3

1:10 I 25.6 21.8 23.7

II 46.8 42.6 42.7
III 16.1 18.9 17.3
Residue 10.2 12.2 14.2
P
98.7 95.5 92.9

1:15 I 28.4 25.1 22.1

II 48.5 41.5 50.6
III 19.1 15.0 15.3
Residue 14.7 14.7 15.0
P
105.7 96.3 103

1:20 I 25.6 22.3 21.2

II 53.7 49.3 46.2
III 17.5 16.8 17.8
Fig. 3. Solubility of collagen after 24 h treatment at 4  C of cod skins Residue 5.4 9.7 15.8
P
with 0.5 M acetic acid solution at different ratios. (^) Whole skins, 105.8 98.1 101
(*) minced skins. Procedure shown in Fig. 1 was used.
 M. Sadowska et al. / Food Chemistry 81 (2003) 257–262 261

in buffer, was exactly the same. This indicates that 15%

of collagen in cod skins contains intermolecular cross-
links, which are stable in acidic media.
Prolonged treatment (72 h) of skins with citric acid
(4:1) did not influence the amount of extracted collagen
(Table 5).
Collagen can be precipitated from solution with
sodium chloride, or with polysaccharides, as well as
freeze-dried. After lyophilisation of collagen solution in
acetic acid, pure collagen can be obtained. However, in
the case of solution in citric acid, collagen is accom-
panied by crystals of citric acid, which can be removed
by dialysis if it is necessary for further applications of
the isolated collagen.
Up to now there is no information concerning differ- Fig. 4. Effect of temperature on the solubility of skin collagen in water
ences in physico-chemical features of native collagen (^) and in 0.45 M NaCl solution (*) at the ratio of minced skins to
extracted from connective tissue with the use of acetic solution of 1:6.
and citric acid. The results of Gómez-Guillén and
Montero (2001) on the influence of organic acid, applied Crodet and Rokafenol N-6, proved to be Rokafenol.
to the extraction of collagen from megrim (Lepi- Citric acid also inhibits the appearance of rancid off-
dorhombus boscii) skins, on the functional features of odour of collagen solutions. The addition of Rokafenol
gelatin, indicate, that the gelling temperature and time N-6 to all extracting solutions, used in the procedure of
are higher and turbidity of gelatin solutions was lower collagen purification and the solubilisation in citric acid,
when citric acid was used for extraction. entirely prevented the development of rancid off-odour
The thermal solubility of collagen depended on the in the preparation when stored for up to 50 days at 0  C.
medium applied (Fig. 4). If cod skins were heated in A similar effect can be achieved at 4  C and at 20  C in
water, the solubility of collagen increased significantly the presence of 0.14% of aseptine M-14 in the collagen
at temperatures above 30  C. At 40  C about 70% of solution in citric acid.
collagen was dissolved. The amount of dissolved col- The method of preliminary chemical treatment of
lagen was larger when the skins were incubated in 0.45 skins from North Atlantic cod applied by Gudmunsson
M NaCl instead in water; it amounted to about 85% at and Hafsteinsson (1997) in the preparation of odourless
30  C. Sodium chloride probably decreased the tem- gelatin, seemed to be very complex and labour-inten-
perature of thermal denaturation of collagen. sive. Washing of skins to pH 7 after numerous extrac-
tions (three times with NaOH, sulphuric and citric
3.3. The influence of storage time on off-odour of acids) was the most time-consuming part of the process.
collagen solutions As a result of such a large number of operations, 25%
of collagen from Baltic cod skins was lost. The proce-
The undesirable feature of collagen extracted from dure was also not effective enough in the preparation of
cod skins with diluted acetic acid is a rancid fish oil odourless collagen.
odour. It can be prevented by preliminary washing of
skins with solutions containing 0.5% detergent. The
most effective detergent in preventing the development 4. Conclusions
of odour, among the detergents tested, i.e. Cetereth,
Skins of Baltic cod contain about 22% collagen. With
Table 5 the use of a one-stage 24-h extraction of the skins with
Effect of time of extraction of Baltic cod skins at 4  C on the con- acetic or citric acid, at a ratio of skins to solvent of 1:6
centration of nitrogen compounds in citric acid extracta
and 1:4, respectively, the same amount of collagen,
Time Skins: Concentration [%] about 20%, can be extracted. In the case of citric acid
[h] solution extraction, the yield of collagen cannot be increased by
Total Crude Hydroxyproline Collagenc increasing the ratio of acid to skins but it can be
nitrogen proteinb
achieved in the case of acetic acid extraction. However,
24 1:4 0.640.005 4.0 0.04 0.26 0.009 3.9 0.14 the collagen solutions in acetic acid are more diluted
72 1:4 0.610.006 3.8 0.04 0.24 0.002 3.5 0.03 and have the characteristic smell of acetic acid. There-
a
Mean valuestandard deviation from three separate samples. fore they are not technically useful. After a single
b
Crude protein=total nitrogen6.25. extraction of skins with acetic acid at a ratio of 1:40, the
c
Collagen=hydroxyproline14.7. collagen concentration in solution amounts to 0.2%,
262 M. Sadowska et al. / Food Chemistry 81 (2003) 257–262

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