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International Journal of Biological Macromolecules 42 (2008) 69

Preparation and partial characterization of collagen


sheet from fish (Lates calcarifer) scales
S. Sankar, S. Sekar, R. Mohan, Sunita Rani,
J. Sundaraseelan, T.P. Sastry
Central Leather Research Institute, Adyar, Chennai 96, India
Received 4 January 2007; received in revised form 17 August 2007; accepted 17 August 2007
Available online 26 August 2007

Abstract
Fish scales, which are hitherto discarded as waste, were collected and cleaned thoroughly. The scales were hydrolyzed under controlled acidic
conditions, neutralized and made in to a sheet, i.e., fish scale collagen sheet (FCS). The FCS was characterized for its infrared spectroscopy (IR),
thermo-gravimetric analysis (TGA), scanning electron microscopy (SEM), and mechanical properties. The IR study has shown that the sheet
contains both organic and inorganic phases revealing that the scales are partially deminaralized. The tensile strength of FCS is enough if it is used
as a wound dressing material. The SEM studies have shown that FCS is porous and exhibited fibrous nature.
2007 Elsevier B.V. All rights reserved.
Keywords: Fish scales; Collagen sheet; Lates calcarifer; Wound dressing material

1. Introduction
Fish scales are composed of connective tissue protein, collagen, covered with calcium salts. The amount of protein ranges
from 41 to 84% and the remaining is calcium phosphate and
calcium carbonate. Several authors extracted collagen and the
inorganic portion from fish scales and characterized the same.
Ikoma et al. [1] extracted type I collagen from fish scales of
Pagrus major and Oreochromis niloticas and studied their physical properties. The enthalpy and entropy estimated from thermal
analysis could be correlated to amino acid sequences (Gly-ProHyp) of type I collagen and the number of methionine and
amino acids residues. Nagai et al. [2] reported that fish scale
collagens were heterotrimers with chain composition of (1)2
and 2 and the denaturation temperature was lower than land
animal collagen. Onozato and Watabe [3] did microscopic investigation of scales of gold fish, Carassius auratus and revealed
that the lamella of fibrillary plates contain sheet-like structures
composed of vertically oriented collagen fibers embedded in
an organic matrix. Zhang et al. [4] determined hydroxyproline
in fish scales using photo colorimetric method. After decalcifi-

Corresponding author. Tel.: +91 44 22582361; fax: +91 44 24911589.


E-mail addresses: sastrytp@sify.com, sastrytp@yahoo.com (T.P. Sastry).

0141-8130/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2007.08.003

cation of fish scales the retaining rate of hydroxyproline was


96.19%. Zylberberg et al. [5] used immunofluorescence and
electron microscopy for analyzing the relationships between
the organization of collagen fibrils in elasmoid scales and the
orientation of microtubules and action microfilaments in the
check scleroblasts producing this collagenous stroma. Tuan et al.
[6] prepared hydroxyapatite (HA) nanoparticles film from fish
scales by high temperature sintering to remove organic component and collect inorganic powder. The pure HAP could be
obtained after pre-treating fish scales in basic solution and calcining at 800 C for 1 h, while only the admix of -TCP and
HAP were obtained by sintering fish scales directly. Duan et
al. [7] extracted gelatin from fish scale, which can be used as
an additive in food, drug and cosmetic fields. The decalcification of the scales was carried out using EDTA method and the
reproducibility and stability show that the method is feasible.
Liu et al. [8] manufactured solution from fish scales for producing functional foods. The solution contains 18 kinds of free
amino acids (2030%) calcium and phosphorus (4.520%), and
trace elements including Mg, Cu, Zn, Mn and Fe (0.151%).
Zhang et al. [9] determined the changes of collagen in the process of decalcification in fish scales. The changes of collagen
and total protein content in hydrochloric acid decalcification of
fish scales were studied. When the concentration of hydrochloric
acid was 0.080.1 mol/L, the content of hydroxyproline colla-

S. Sankar et al. / International Journal of Biological Macromolecules 42 (2008) 69

gen was highest. Yamauchi et al. [10] prepared multi-layered


sheets (210 layers), which consisted of alternatively accumulated collagen as calcium phosphate layers with thickness
of 68 m in each layer. The inorganic layer was mineralized by means of alkaline phosphatase-catalyzed hydrolysis of
water-soluble phosphate esters in the presence of calcium ions.
The enzymatic mineralisation and collagen/calcium phosphate
composites sheets were discussed in conjugation with physiochemical and biological properties.
In the present study we report the preparation of collagen
sheet from the fish scales and its characterization.
2. Materials and methods
2.1. Materials
Fig. 1. Collagen sheet from fish scales.

Fish scales were isolated from fresh fish of sea catch and
washed thoroughly in running water and dried at 40 C in a hot air
oven, stored in polythene bag at room temperature (34 2 C)
for 24 h. All other reagents used in this study were of analytical
grade.

2.4. Infrared spectroscopy


IR spectroscopy of fish scale was taken using Nicolet Impact
400 FTIR spectroscopy by preparing a 500 mg KBr pellet containing 26 mg of the sample.

2.2. Methods
2.5. Thermal gravimetric analysis
Dry fish scales were treated with different concentrations
of HCl solutions (1:1, v/w, water/fish scales; i.e., 100 ml
water/100 g fish scales; Table 1) for 24 h at room temperature
(34 2 C). Later, acid solution was decanted and the scales
were washed with water. Then water was added to scales (2:1,
v/w) and pH of the scales was adjusted to 7 using 0.1N NaOH
solution (to check the pH a sample scale was cut vertically,
pH paper was inserted and the pH was noted). Further, scales
were washed with water and pulverized with a float of 1:1 (v/w,
water/scales) using a domestic mixer for 15 min at 12,000 rpm
(the scales treated with 2.81N HCl solution could be pulverized
and made into paste), resulting paste is cast into sheet in a polythene tray and dried at room temperature (34 2 C). The dried
sheets are stored in polythene covers for further studies (Fig. 1).

The thermal stability was determined with a thermogravimetric (TG) analyzer (Perkin-Elmer TGA) over a
temperature range of 37585 C at a heating rate of 20 C/min
under nitrogen atmosphere.
2.6. Scanning electron microscopy
The sample was coated with gold ions using an ion coater
(fisons sputter coater) under the following condition: 0.1 Torr
pressure, 20 mA current, and 70 s coating time. Surface structure was visualized by scanning electron microscope (JSM 5300
Scanning microscope) using a 15 kV accelerating voltage.
2.7. Shrinkage temperature

2.3. Tensile strength


Two dumbbell-shaped specimens of 4 mm wide and 10 mm
length were punched out from the prepared films using a die.
Mechanical properties such as tensile strength (MPa) and percentage of elongation at break (%) were measured using a
universal testing machine (INSTRON model 1405) at an extension rate of 5 mm/min.

Shrinkage temperature was measured by observing dimensional changes in individual fibers exposed to solvent media by
using a traveling microscope filled with a hot stage [11] a portion of dry collagen was shaken with excess of solvent media.
The wetted fibers were placed to the center of a ring of light
on a microscope slide and sealed with a cover slip. The temperature was raised at a controlled rate of 0.51 C/min. and the
temperature of incipient of dimensional changes noted.

Table 1
Effect of HCl solution on fish scales

3. Results

Concentration

Observation

20% HCl solution (2.24N)


25% HCl solution (2.81N)

No effect on scales
Scales could be made into a paste
and cast into a sheet
Scales were dissolved

30% HCl solution (3.37N)

The IR spectrum (Fig. 2) of fish scale collagen has shown


amide peaks at 1676 and 1255 cm1 , which represent amide-I
and -III, respectively, and obviously amide-II peak is absent.
Peaks at 1038 and 1176 cm1 represent PH bending and
phosphate stretching, respectively, indicating the presence of

S. Sankar et al. / International Journal of Biological Macromolecules 42 (2008) 69

Fig. 2. IR spectrum of FCS.

calcium salts. The prominent peak at 2927 cm1 and shoulder at


2861 cm1 represent the CH2 CH3 stretching vibrations, which
are characteristic for collagen.
Thermal decomposition profile of FCS is shown in (Fig. 3).
In this investigation, FCS is heated at steadily increasing temperature from 30 to 650 C. An initial weight loss of 15.51% was
observed, due to loss of water up to 120 C. Second major weight
loss was observed between 249 and 338 C with maximum
weight loss of 51.1% at 293 C. The third weight loss (33.5%)
occurred between 535 and 635 C with maximum weight loss
at 592 C. The tensile strength of FCS prepared was found to be
2 MPa.
SEM images of collagen sheet (Figs. 4 and 5) have shown
fibrous and porous nature. As the mineral present in the scale
was partially dissolved by acid, porous nature of the sheet is
clearly sheen. The fibrillar structure of collagen present in the
sheet and its organization are clearly viewed.

Fig. 4. Scanning electron micrograph of FCS (300).

observed in bovine collagen at around 15501560 cm1 is missing in the FCS IR spectrum. The PH bending and phosphate
stretching bands indicate that there is inorganic phase in the FCS,
this obviously shows that fish scales were partially demineralized.
In thermo-gram the losses of weight due to evaporation of
water, CO, CO2 and evaporation of other pyrolysis products are
collectively measured as percentage of original weight. Initial
weight loss of 15.5% up to 120 C is due to the loss of water.
The second weight loss (between 249 and 338 C) of 51.1% is

4. Discussion
The amide-I and -III peaks represents the protein portion,
i.e., collagen in the FCS, but amide-II band which is normally

Fig. 3. Thermo-gram of FCS.

Fig. 5. Scanning electron micrograph of FCS (300).

S. Sankar et al. / International Journal of Biological Macromolecules 42 (2008) 69

mainly due to the decomposition of protein present in the sheet.


This type of protein loss was observed in our earlier studies
[12]. The third weight loss of 33.5% between 535 and 635 C
is attributed to the loss of inorganic phase in FCS. This thermo
gram revels that FCS is a two-phase system containing protein
and inorganic (calcium phosphate) phase.
The main objective of the preparation of FCS is to use it as a
wound dressing material. Pure collagen was not isolated in this
study. The fish collagen sheet contains collagen and mineral. As
it is in sheet form we have estimated the shrinkage temperature
of fish scales and FCS to correlate the dimensional changes in
the individual fibers. It was found that the shrinkage temperature
of fish scales was 45 1 C and FCS 39 1 C. Hence drying at
room temperature 34 2 C shall not denature the FCS fibers.
Fish scales as such have very high tensile strength (90 MPa)
because of the hierarchically ordered structure of mineralized
collagen fibers. In contrast, demineralized scales have significantly lower tensile strength (36 MPa), indicating that the
interactions between the apatite crystals and collagen fibers are
of fundamental importance in determining the mechanical properties [13]. The tensile strength of FCS prepared was found to
be 2 MPa. This strength is very low as the deminerlized scales
were ground into a paste and reconstituted into a sheet and naturally, the reconstituted sheets will have poor tensile strength.
The tensile strength of he collagen sheet prepared is sufficient
to handle when it is used as a wound dressing material [14].
The FCS is proposed to be used on wound dressing material on leprosy, diabetic and other chronic ulcers. As the use of
bovine collagen may be restricted in future due to mad cow and

foot and mouth diseases, the collagen from other sources have
to be tried as wound dressing materials and for other pharmaceutical uses. In this direction FCS may be an alternative to the
bovine collagen. The porous nature of sheet will help to absorb
the wound fluid when it is applied on wound and thereby keeping wound dry and this property helps in enhancing the rate of
healing of the wound.
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