Vol.3, No.1B, 11-15 (2014) Journal of Agricultural Chemistry and Environment
Copyright © 2014 SciRes. OPEN ACCESS
The calcium-binding activity of fish scale protein
Ruiyan Nie, Yuejiao Liu, Zunying Liu#
College of Food Science and Engineering, Ocean University of China, Qingdao, China;
#Corresponding Author: liuzunying@ouc.edu.cn.
Received September 2013
The calcium-binding activity of tilapia scale pro-
tein hydrolysates sequentially hydrolyzed by
trypsin, fl avo r enzyme and pepsin were investi-
gated. The hydrolysates were divided into four
fractions using G-15 gel chromatography, and
the F3 fraction has the higher calcium-binding
activity of 196.3 mg/g. The UV-vis and the
Fourier transform infrared spectroscopy (FTIR)
demonstrate that the amino nitrogen atoms and
the oxygen atoms belonging to the carboxylate
groups are the primary binding sites for Ca2+.
The X-ray diffraction and scanning electron mi-
croscopy (SEM) confirmed the reaction between
the peptde and calcium. The results obtained
indicated that this fish scale protein hydroly-
sates have potential as functional foods for cal-
Tilapia; Fish Scale; Calcium-Binding Activity;
Calcium (Ca2+) is the fifth most abundant element in
the earth’s crust and the most abundant cation in the hu-
man [1]. The ionic form of Ca2+ serves as a universal
intracellular messenger to modulate many processes such
as neurotransmission, enzyme and hormone secretion,
cell cycle regulation and programmed cell death [2,3].
The major source of calcium was provided by diet, espe-
cially milk and dairy preparations. However, calcium
deficiency is widespread due to insufficient intake and
diminished solubility caused by other constituents in
food, like phytates, cellulose, fats, etc. [4,5]. Calcium de-
ficiency causes hypocalcemia, bone mass loss and in-
crement in the risk of osteoporosis development either in
humans [6]. Therefore it is crucial to prevent calcium
deficiency by regulating the calcium absorption and cal-
cium solubility.
Casein phosphopeptides (CPP) enhancing the absorp-
tion of calcium has been observed by some investigators
[7,8]. However, the relatively high price and lactose in-
tolerance of CPP inevitably limits its universal applica-
tion on calcium supplement. Consequently, exploiting
novel calcium compositions based on other protein hy-
drolysates is an ideal alternative, such as hen egg yolk
phosvitin, fish bone and soy protein [9-11]. Nile tilapia
(Oreochromisniloticus ) was widely distributed, and its
annual aquaculture production in 2010 was 2.54 million
tons according to the statistics of Food and Agriculture
organization of United Nations [12]. However, the ma-
jority of the scales were dumped during the fish
processing and damaged to the environment. Fish scale is
rich in protein and calcium, therefore it is an ideal re-
source to produce calcium supplement as alternatives of
CPP. The objective of this study was to evaluate calcium-
binding activity of fish scale protein hydrolysates and the
physical properties of pe pt ide-calcium complex.
2.1. Materials
Nile Tilapia fish scales were obtained from Shandong
Meijia Group (Rizhao, China). Pepsin (porcine gastric pep-
sin, activity 20 unitsmg1 protein) was provided by Si-
nopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Trypsin (powder, porcine 1:250 > 250 USP unitsmg1),
Flavourzyme (from Aspergillusoryzae, >20 unitsmg1),
and crystalline bovine albumin, Sephadex G-15 were
purchased from Sigma Chemical Co. (Sigma-Aldrich
Inc., St. Louis, MO, USA). All other chemicals were of
analytical reagent grade.
2.2. Preparation of Fish Scale Protein
The fresh scale was stirred in 1.5 M NaCl solution for
*This research was sup ported by the National Natu ral Science Founda-
tion of China (No. 31101379).
R. Y. Nie et al. / Journal of Agr i cu l tural Chemi stry and Environment 3 (2014) 11-15
Copyright © 2014 SciRes. OPEN A CCESS
24 h and then decalcified with 0.4 M HCl. The scale was
washed by deionized water (the conductivity was about 1
- 1.5 µScm1), then dried and smashed to powder (sieved
by 80 mesh sieve). The mixture of fish scale powder and
deionized water (powder:water, W:W = 1:20) was hy-
drolyzed by pepsin (1.5%, W/W) at pH 1.8, 40˚C for 5 h.
Then 0.75% trypsin (w/w) was added for 5 h hydrolysis
after pepsin was inactiviated and the pH was neutralised
to 8.0. The sample was boiled for 10 min and adjusted to
pH 7.0, and then flavourzyme (0.75%, w/w) was con-
ducted further hydrolysis at 50˚C for 5 h before it was
inactivated. At various time of hydrolysis, an aliquot hy-
drolysate was collected for calcium-binding capacity de-
termination. The mixture and collected aliquot were cen -
trifuged at 3000 × g for 20 min at 4˚C, filtered through
0.45 μm Millipore filters to collect soluble pep tides. The
peptides was freeze-dried and analyzed. The experiment
was triplicated.
2.3. Isolation of Calcium-Binding Peptide by
Sephadex G-15
Demineralized fish scale protein hydrolysate was
loaded into Sephadex G-15 (Pharmacia, NJ, USA) on an
open column (2.6 × 70 cm) at the flow rate of 0.6
mLmin1. Fractions corresponding to major peaks were
collected and lyophili zed immediately. All the process of
chromatography was monitored at 280 nm according to
the method of Jung et al. [13].
2.4. Preparation of Peptide-Calcium
The binding reaction was performed by mixing fish
scale protein hydrolysates at the concentration of 10%
(w/w) with Ca2+ at the concentration of 60 mmo lL1
under continuous stirring for 30 min at 50˚C. Thereafter,
the free calcium was removed with a 100 Da molecular-
weight cut -off semi-permeable membrane (Thermo
Fisher Scientific Inc., Waltham, UK). The retentate was
collected and lyophilized for further analysis.
2.5. Calcium-Binding Activ ity Analysis
Calcium-binding capacity was measured following the
method reported in a previous study [14]. Demineralized
samples with maximum concentration of 1000 mgL1
were mixed with 20 mM sodium phosphate buffer (pH
7.8) and 5 mM CaCl2. Then the mixture was incubated at
22˚C for 30 min with continuous stirring under pH 7.8.
Calcium phosphate precipitate was removed by filtrated
through a 0.45 μm membrane, the calcium content of the
filtrate was assayed by flame atomic absorption spec-
troscopy. The experiment was triplicated and means were
2.6. Fourier Transform Infrared (FTIR) and
UV-Visible Spectroscopy
FTIR spectra were obtained using discs containing 0.2
mg of calcium-binding peptide or peptide-calcium com-
plex mixed with 20 mg dried KBr powder. The spectra
were recorded using an infrared spectrophotometer (Ni-
colet 200SXV, Thermo-Nicolet Co., Madison, WI, USA)
from 4000 to 400 cm1 at a data acquisition rate of 4
cm1 per point. The peak signals in the spectra were ana-
lysed using Omnic 6.0 software (Thermo-Nicolet Co.,
Madison, WI, USA). The absorption spectra were also
recorded in the 200 - 400 nm region with a Shimadzu
spectrophotometer (Model UV-2550 PC) using distilled
water as a reference.
2.7. X-Ray Diffraction (XRD) Analysis
XRD patterns of calcium-binding peptide and the
complex were obtained using an X-ray diffractometer
(Model D/MAX 2500, Rigaku International Corporation,
Japan) with Cu radiation (λ = 1.54 Å) at 40 kV and 40
mA. Samples were scanned from 2θ = 4˚ - 90˚ at a scan -
ning rate of 4˚/min. The gallery height (d-spacing dis-
tance) was determined by the peak in the XRD pattern
and expressed by Bragg’s equation (λ = 2sinθ).
2.8. Microstructure of Calcium-Binding
Peptide and the Complex
The microstructure of calcium-binding peptide and the
complex were performed by a scanning electron micro-
scope (JSM-840, JEOL Tokyo, Japan). The powder sam-
ples were sprayed and sputter-coated with gold (Sputter
coater SPI-Module, PA, USA). The specimens were ob-
served at an acceleration voltage of 25 kV.
2.9. Statistical Analy sis
Experiments were condu cted in triplicate. Comparison
of means was performed by Duncan’s test with confi-
dence level as P ≤ 0.05.
3.1. Isolation of Calcium-Binding Peptide
Tilapia scale protein hydrolyzates were separated into
a Sephadex G-15 column and four major fractions (F1,
F2, F3, F4) were eluted at different retention time based
on their molecular weights (Figure 1). Of which, F3
fraction with higher calcium-binding activity of 196.3
mg/g protein was collected for further analysis (Table 1).
3.2. F TIR Analysis
The FTIR spectra of the calcium-binding peptide and
R. Y. Nie et al. / Journal of Agr i cu l tural Chemi stry and Environment 3 (2014) 11-15
Copyright © 2014 SciRes. OPEN ACCESS
Tabl e 1. The calcium-binding activity of the fractions.
Fractions Calcium-binding activity
(mg Calcium/g protein)
Crude hydrolysates 93.6 ± 3.5c
F1 fractions 48.0 ± 0.2f
F2 fractions 63.3 ± 1.1d
F3 fractions 196.3 ± 1.8b
F4 fractions 52.1 ± 1.4e
Casein phosphopeptides 446.0 ± 1.2a
Figure 1. G-15 gel chromatography of fish scale protein hy-
the peptide-calcium complex were shown in Figure 2.
Several absorption bands for the calcium-binding peptide
in the range 525 to 849 cm1 and 1034 to 1163 cm1 arise
from the vibration of the C-H and N-H bonds [15], and
these bands were not present in the peptide-calcium com-
plex spectra. The band (1419 cm1) for the -COO car-
boxylate group moved to a lower frequency (1406 cm1)
in the spectrum of the peptide-calcium complex, and
these vibrations were also observed by Reddy et al. [16]
when metal ions bind to the amino acids residues in pep-
tides, indicating the calcium binds to the fish scale pep-
tide primarily through interactions with carboxyl oxygen
and amino nitrogen atoms .
3.3. UV Scanning Analysis
As can be seen in Figure 3, strong absorption ap-
peared near 200 nm and 280 nm, which could be ex-
plained by the spectral characteristics of the peptide bond
and aromatic amino acids residues in the peptide [17]. In
addition, the absorption intensity for the peptide-calcium
complex and CaCL2 is somewhat lower in the near ultra-
violet region when compared with absorption intensity
for the peptide alone, indicating calcium was bound by
the fish scale peptide. The results were also proved by
Figure 2. FTIR spectra of the calcium-binding peptide (A) (F3
Fraction) and t he peptide-calcium complex (B).
Figure 3. UV-vis absorption spectrum of the calcium-binding
peptide (A) (F3 Fraction), the peptide-calcium complex (B) and
Armas et al. [18] and Jin et al.[19].
3.4. X-Ray D ifferaction Analysis
The X-ray diffractograms of the peptide and peptide-
calcium complex is shown in Figures 4(A) and (B). The
diffractogram of the peptide shows two diffraction peaks
at approximately 7˚ - 8˚ and 21˚ - 23˚. The intensity of
the diffraction peak at 7˚ - 8˚ in the peptide-calcium com-
plex becomes sharp and narrow, illustrating that the pres-
ence of calcium increases the crystallinity of the complex.
This phenomenon is due to the significant interaction
between peptide amino acid and calcium [19].
3.5. M icrostructure Analysis
The electron micrographs of peptide and peptide-cal-
cium complex were obtained in multiples of 10,000 (F ig-
ure 5). The peptide shows a smooth amorphous structure,
however the peptide-calcium complex shows a more fold,
crystal and multi-branched structure than that of the pep-
tide. This result could be due to the interaction between
R. Y. Nie et al. / Journal of Agr i cu l tural Chemi stry and Environment 3 (2014) 11-15
Copyright © 2014 SciRes. OPEN A CCESS
Figure 4. X-ray differaction of the calcium-binding
peptide (A) (F3 Fraction) and the peptide-calcium
complex (B).
Figure 5. Microstructure of the calcium-
binding peptide (A) (F3 Fraction) and the
peptide-calcium com pl ex (B).
the peptide and the calcium, and this might be associated
with the sharper and narrower diffractograms of peptide-
calcium complex when calcium was added (Figure 4).
The results of this study showed that enzymatic hy-
drolysis was the effective way to recover the calcium-
binding peptide form fish scales. The F3 fraction sepa-
rated by Sephadex G-15 chromatography possessed
higher calcium-binding activity. The FTIR spectrum of
the peptide-calcium complex indicated that the calcium
interacts with the fish scale peptide, mostly via amino
nitrogen atoms and the oxygen atoms of carboxylate
groups. The UV-vis spectra, X-ray diffractogram and the
microstructu re of the peptide-calcium complex con-
firmed the changes of the peptide after adding the cal-
cium, indicating the interaction between the peptide and
calcium. These results indicating fish scale protein hy-
drolysates possess the huge potential to be an alternative
for CPP as calcium supplement.
[1] Peng, J.B., Brown, E.M. and Hediger, M.A. (2003) Apic-
al entry channels in Ca2+ transporting epithelia. News in
Physiological Science, 18, 158-163.
[2] Clapham, D.E. (1995) Intracellular Ca2+ replenishing the
stores. Nature, 375, 634-635.
[3] Berridge, M.J. (1995) Capacitative Ca2+ entry. Biochemi-
cal Journal, 312, 1-11.
[4] Reinhold, J.G., Lahimgarzadeh, A., Nasr, K. and Hedaya-
ti, H. (1973) Effects of purified phytate and phytate-rich
bread upon metabolism of zinc, calcium, phosphorus, and
nitrogen in man. The Lancet, 301, 283-288.
[5] Slavin, J.L. and Marlett, J.A. (1980) Influence of refined
cellulose on human bowel function and calcium and mag-
nesium balance. The American Journal of Clinical Nutri-
tion, 33, 1932-1939.
[6] Centeno, V.A., Barboza, G.E.D., Marchionatti, A.M.,
Alisio, A.E. Dallorso, M.E., Nasif, R. and Talamoni,
N.G.T. (2004) Dietary calcium deficiency increases Ca2+
uptake and Ca 2+ extrusion mechanisms in chick entero-
cytes. Comparative Biochemistry and Physiology, Part A,
139, 133-141.
[7] Lee, Y.S., Noguchi,T. and Naito, H. (1979) An enhanced
intestinal absorption of calcium in the rat directly attri-
buted to dietary casein. Agricultural Biology and Chemi-
stry, 43, 2009-2011.
[8] Sato, R., Noguchi, T. and Naito, H. (1986) Casein phos-
R. Y. Nie et al. / Journal of Agr i cu l tural Chemi stry and Environment 3 (2014) 11-15
Copyright © 2014 SciRes. OPEN ACCESS
phopeptide (CPP) enhances calcium absorption from the
ligated segment of rat small intestine. Journal of Nutri-
tional Science and Vitaminology, 32, 67-76.
[9] Choi, I., Jung, C., Choi, H., Kim, C. and Ha, H. (2005)
Electiveness of phosvitin peptides on enhancing bioavai-
lability of calcium and its accumulation in bones. Food
Chemistry, 93, 577-583.
[10] Gómez-Guillén, M.C., Giménez, B., López-Caballero,
M.E. and Montero, M.P. (2011) Functional and bioactive
properties of collagen and gelatin from alternative
sources: A re view . Food Hydrocolloids, 25, 1813-1827.
[11] Bao, X.L., Song, M., Zhang, J., Chen, Y. and Guo, S.T.
(2007) Calcium-binding ability of soy protein hydroly-
sates. Chinese Chemical Letters, 18, 1115-1118.
[12] Food and Agriculture Organization (FAO) (2012) Fishery
and Aquaculture Statistics. In: FAO Yearbook, FAO, Rome,
[13] Jung, W.K. and Kim, S.K. (2007) Calcium-binding pep-
tide derived from pepsinolytic hydrolysates of hoki (Joh-
nius belengerii) frame. European Food Research and
Technology, 224, 763-767.
[14] Sato, R., Shindo, M., Gunshin, H., Noguchi, T. and Naito,
H. (1991) Characterization of phosphopeptide derived
from bovine β-casein: An inhibitor to intra-intestinal pre-
cipitation of calcium phosphate. Biochimica Biophysica
Acta, 1077, 413-415.
[15] Silverstein, R.M., Bassler, G.C. and Morrill, T.C. (1981)
Spectrometric identification of organic compound. 4th
Edition, Wiley, New Yor k, 100-180.
[16] Reddy, P., Radhika, M. and Manjula, P. (2005) Synthesis
and characterization of mixed ligand complex of Zn (II)
and Co (II) with amino acids: Relevance to zinc binding
sites in zinc fingers. Journal of Chemical Sciences, 117,
[17] Goldfarb, A.R., Saidel, L.J. and Mosovich, E. (1951) The
ultraviolet absorption spectra of proteins. The Journal of
Biological Chemistry, 193, 397-404.
[18] Armas, A., Sonois, V., Mothes, E., Zazarguil, H. and Fal-
ler, P. (2006) Zinc binds to the neuroprotective peptide
humanin. Journal of Inorganic Biochemistry, 100, 1672-
[19] Jin, Y.G., Fu, W.W. and Ma, M.H. (2011) Preparation and
structure characterization of solublebone collagen peptide
chelating calcium. African Journal of Biotechnology, 10,
doi: 10.5897/AJB10.1923