journal of functional foods 10 (2014) 46–53

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A specific peptide with calcium chelating

capacity isolated from whey protein hydrolysate

Lina Zhao a,b,☆, Shunli Huang a,☆, Xixi Cai a, Jing Hong a,
Shaoyun Wang a,*
a
College of Bioscience and Biotechnology, Fuzhou University, Fuzhou 350002, China
b
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China

A R T I C L E I N F O A B S T R A C T

Article history: A specific peptide displaying calcium-binding capacity was purified from whey protein hy-
Received 21 February 2014 drolysate. The isolation procedures included DEAE anion-exchange chromatography, Sephadex
Received in revised form 14 May G-25 gel filtration, and reversed-phase high-performance liquid chromatography (RP-
2014 HPLC). The amino acid sequence of the peptide was determined to be Phe-Asp (FD), using
Accepted 16 May 2014 liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/
Available online MS). The calcium binding capacity of FD reached 73.34 μg/mg, and the amount increased
by 116% when compared to the whey protein hydrolysate complex. The structural proper-
Keywords: ties of the purified peptide were identified using fluorescence spectra, Fourier transform in-
Whey protein hydrolysate frared spectroscopy (FTIR), and 1H nuclear magnetic resonance (NMR) spectroscopy, respectively.
Calcium-chelating peptide The results indicated that the amido and carboxy groups of the purified peptide were trans-
Purification formed during chelation. The oxygen atoms of the carboxy group and the nitrogen atoms
Structural property of the amido group could chelate calcium to form coordinate bonds by donating electron
pairs. Furthermore, FD-Ca chelate was found to be more stable and absorbable than CaCl2
under both acidic and basic conditions. Our findings suggest that the purified dipeptide Phe-
Asp has the potential to be used as a calcium-binding ingredient in dietary supplements.
© 2014 Elsevier Ltd. All rights reserved.

area of research focus all over the world. Enzymatic modifi-

1. Introduction
Whey protein is a mixture of globular proteins isolated from
whey, the liquid material created as a by-product of cheese
production (Madureira, Pereira, Gomes, Pintado, & Malcata,
2007). With the increased production of cheese from milk,
more and more whey protein was released, the reasonable
exploitation of whey protein becomes particularly significant
(Siso, 1996). However, whey protein is a kind of sensitive pro-
teins whose solubility decreases in acid or heat conditions.
The modification of whey protein has become an immediate
cation can produce biological active peptides. As one kind of
bioactive substances in functional foods, enzyme-hydrolyzed
whey peptides have garnered increased attention in recent
years, the effects of hydrolyzed whey peptides on human
health are of great interest and are currently being investi-
gated as a way of reducing health risk, as well as a possible
supplementary treatment for several diseases (Clemente, 2000;
Kim et al., 2007a; Théolier, Hammami, Labelle, Fliss, & Jean,
2013).
Calcium deficiency results in hypertension, osteoporosis and
intestine cancer (Osborne et al., 1996). The intake of calcium

☆
Co-first authors.
* Corresponding author. Tel.: +86 591 22866375; fax: +86 591 22866278.
E-mail address: shywang@fzu.edu.cn (S.Y. Wang).
http://dx.doi.org/10.1016/j.jff.2014.05.013
1756-4646/© 2014 Elsevier Ltd. All rights reserved.
 journal of functional foods 10 (2014) 46–53
could increase the bone density in children and it is essen-
tial among the middle-aged and the aged to prevent osteopo-
rosis (Cilla et al., 2011; Guénguen & Pointillart, 2000). With the
increase in population of the aged throughout the world, there
is a growing interest in developing calcium supplementary
medicine to prevent and treat bone disease (Kim & Lim, 2004).
The ionized calcium has served as main calcium supple-
ments for human beings in recent years (Lee & Song, 2009).
However, the disadvantage of ionized calcium is that it is prone
to form calcium phosphate deposition in basic intestine en-
vironment (Bronner & Pansu, 1998). As a result, the
bioavailability of dietary calcium is severely lowered. The organic
calcium supplement including calcium-binding peptides has
been becoming one of popular research topics (Narin, Benjamas,
Nualpun, & Wirote, 2013). Hydrolyzed whey peptides, ob-
tained from proteolytic digestion, have shown the consider-
able capacity in incorporating with divalent ions such as
calcium, iron ion, etc. (Chaud et al., 2002; Kim et al., 2007b).
The chelating complex chelated between whey peptides and
calcium ion can promote calcium absorption in human body
and therefore improve its bioavailability.
The objective of this study was to purify and characterize
a highly specific calcium-binding peptide from whey protein
hydrolysates. Whey protein was herein hydrolyzed, a specific
calcium-binding peptide was purified, and mechanism of action
was investigated. The finding would be of significance in uti-
lizing the hydrolyzed peptides from whey protein as calcium-
binding peptide ingredients in functional foods.
2. Materials and methods
2.1. Reagents and materials
Whey protein was purchased from Hilmar Corporation (Batch
No. 20111107) (USA). Flavourzyme (2000 U/mg) and Protamex
(1500 U/mg) were obtained from Novo (Novozymes, Denmark).
Toyopearl DEAE-650M and Sephadex G-25 were purchased from
Amersham Pharmacia Co. (Uppsala, Sweden). All reagents and
chemicals were of analytical reagent and high-performance
liquid chromatography (HPLC) grade.
2.2. Preparation of whey protein hydrolysates
Whey protein solution 5% (w/v) was denatured at 80 °C for
20 min, then the pH was adjusted to 7.0. The sample was hy-
drolyzed using Flavourzyme and Protamex (2:1, w/w) with a
substrate:enzyme ratio of 25:1 (w/w) at 49 °C for 7 h. Hydro-
lysate was terminated by heating the sample in boiling water
for 10 min to inactive the enzyme. The mixture was cooled to
room temperature and subsequently centrifuged at 16,000 × g
for 20 min. The supernatant, referred to as whey protein hy-
drolysate (WPH), was lyophilized and stored at −20 °C for sub-
sequent purification.
2.3. Purification of calcium-binding peptide
A slurry of Toyopearl DEAE-650M was packed in a column
(20 × 2.5 cm), then equilibrated with 5 column volume (CV) of
47
equilibrating buffer and 20 mM Tris-HCl buffer (pH 9.0). After-
wards, 100 mg of lyophilized hydrolysates that had been through
the 0.45 μm filter film was dissolved in 10 mL of the same buffer
(pH 9.0) and loaded on the column. The column was washed
with equilibrating buffer, the collected peak was labeled as the
non-absorbed fraction. The bound peptides were eluted using
a gradient elution with the same buffer containing 0–0.5 M NaCl
at a flow rate of 0.5 mL/min and fraction volume was 5 mL/
tube. Elution was monitored by measuring the absorbance at
214 nm. The calcium-binding abilities of all fractions were de-
termined. The peak exhibiting the strongest ability was col-
lected for the subsequent isolation.
The sample (200 mg) exhibiting the strongest binding ability
from DEAE was dissolved in 5 mL deionized water and loaded
onto a Sephadex G-25 column (100 × 2.0 cm), which had been
previously equilibrated with deionized water, it was eluted with
deionized water at the flow rate of 0.3 mL/min. Elution was
monitored by measuring the absorbance at 214 nm. After
calcium-binding capacity was determined, the fraction with
the highest activity was pooled and lyophilized.
The lyophilized sample collected from the G-25 column was
dissolved in approximately 30 mg/mL distilled water and pu-
rified by semi-preparation reversed-phase (RP)-HPLC on a C18
reversed-silica gel chromatograph (Gemini 5 μ C18, 250 × 10 mm;
Phenomenex Inc.; Torrance, CA, USA). The injection volume was
200 μL. Elution was performed with solution A (0.05%
trifluoroacetic acid (TFA) in water) and solution B (0.05% TFA
in acetonitrile) with a gradient of 0–30% B at a flow rate of
4.0 mL/min for 50 min. The elution was monitored at 214 nm,
and also collected for calcium binding capacity analysis. The
most active fraction was chosen for analytical HPLC analysis.
Further purification was performed using an analytical C18
column. Buffers A and B were the same as those used in prepa-
ration for RP-HPLC. Runs were conducted with a liner gradi-
ent of 0–10% solvent B at a flow rate of 1 mL/min. All eluted
peaks were monitored at 214 nm.
2.4. Peptide identification by mass spectrometry
The purified peptide was analyzed using a liquid
chromatography/electrospray ionization (LC/ESI) tandem mass
spectrometer (Delta Prep 4000; Waters Co.; USA) from 300 to
3000 m/z.
2.5. Calcium-binding activity assay
The lyophilized sample was dissolved in deionized water to
a final concentration of 1.0 mg/mL. The peptide solution was
mixed with the solution containing excessive CaCl2 (5 mM) and
superfluous 0.2 M sodium phosphate buffer (pH 8.0). The so-
lution was stirred at 37 °C for 2 h and the pH was maintained
at 8.0 using a pH meter. The calcium-binding peptide could
inhibit formation of insoluble calcium phosphate through com-
petitively combining with CaCl2. The reaction mixture was cen-
trifuged at 10,000 × g at room temperature for 10 min in order
to remove insoluble calcium phosphate salts. The calcium
content of the supernatant was determined using a colori-
metric method with ortho-cresolphthalein complexone reagent
(Gitelman, 1967). The absorbance at 570 nm was determined
after adding the working solution to the sample. All experi-
48 journal of functional foods 10 (2014) 46–53
ments were performed in triplicate, and the values were ex-
pressed as mean ± standard deviation (SD).
2.6. Structural characterization of peptide–calcium
complex
2.6.1. Preparation of peptide–calcium complex
The calcium-binding peptide was prepared by adding 5 mL of
1% (w/v) CaCl2 to 20 mL of 2.5% (w/v) calcium-binding peptide
solution. The reaction was placed in a controlled water bath
with constant agitation (100 rpm) at 37 °C for 2 h after the pH
of the solution was adjusted to 7.0 by the addition of 0.1 M
NaOH. Absolute ethanol (nine times volume of the solution)
was added to the solution to remove free calcium, the mixture
was centrifuged at 10,000 × g for 10 min, and the precipitate
was lyophilized for analysis.
2.6.2. Fluorescence spectra
Fluorescence spectra were measured to monitor conforma-
tional changes in the peptide induced by calcium chelation
using a Hitachi F-4600 fluorescence spectrophotometer (Hitachi
Co.; Japan). The excitation wavelength was 295 nm and emis-
sion wavelengths between 310 and 400 nm were recorded.
2.6.3. Fourier transform infrared spectroscopy (FTIR)
Freeze-dried sample (1 mg) mixed with 100 mg of dried KBr was
loaded on the FTIR spectrograph. All FTIR spectra were re-
corded using an infrared spectrophotometer from 4000 to
400 cm−1 (360 Intelligent; Thermo Nicolet Co.; USA). The peak
signals in the spectra were analyzed using OMNIC 8.2 soft-
ware (Thermo Nicolet Co.; Madison, WI, USA).
1 sessing the highest calcium-binding capacity, was isolated on
2.6.4. H nuclear magnetic resonance spectroscopy (NMR)
The peptide–calcium complex and the peptide (0.5 mg) were
dissolved in 500 μL deionized water. Fifty microliters of deu-
terium oxide (D2O) was added after the pH of the solution was
adjusted to 6.5. The samples were transferred into 5 mm NMR
tubes and subjected to NMR analysis with a Bruker Avance III
spectrometer (Bruker Biospin; Rheinstetten, Germany).
2.6.5. Calcium releasing percentage of peptide–calcium
complex
The calcium-releasing percentage of peptide–calcium complex
and CaCl2 were assayed at various pH values, including 2.0, 3.0,
4.0, 5.0, 6.0, 7.0, and 8.0. After incubation in a shaking water
bath at 37 °C for 2 h, the solutions were centrifuged in a re-
frigerated centrifuge at 10,000 × g for 10 min (Wang, Zhou, Tong,
& Mao, 2011). The calcium content of the supernatant and the
total calcium in the solution were measured using a colori-
metric method with ortho-cresolphthalein complexone reagent.
The calcium-releasing percentage was calculated as follows:
Calcium-releasing %
= Calcium in supernatant total calcium in solution × 100%
2.7. Statistical analysis
All experiments were determined in triplicate. Statistical analy-
sis was performed using the SPSS software program (SPSS Inc.;
Chicago, IL, USA). Data were presented as mean ± standard de-
viation (SD). Statistical significance was determined by one-
way analysis of variance (ANOVA) followed by Duncan’s multiple
range test. Statistical analysis was performed using the soft-
ware Origin 8.0 (Origin Lab Co.; USA).
3. Results and discussion
3.1. Purification of calcium-chelating peptide
The eluted fractions separated from DEAE anion exchange chro-
matography column was shown in Fig. 1A. Calcium-binding
capacity of all eluted fractions was determined accordingly,
and the fraction with the highest calcium-binding activity was
marked as F2 (Fig. 1B). Previous literatures suggested that
whether the peptide combined with metal ion mainly
depended on the net charge of the amino acids or peptides
(Chaud et al., 2002; Reddy & Mahoney, 1995). Fraction 2 (F2 in
Fig. 1A), which was negatively charged in the pH 9.0 condi-
tion of the equilibration buffer, showed greater affinity to
positively charged calcium. Differences in the net charge
seemed to influence their calcium-binding activities (Chaud
et al., 2002).
Chaud et al. (2002) reported that the length of peptides could
influence their metal ion binding activities. On G-25 gel filtra-
tion chromatograph column, size-dependent fractionation of
the samples was shown in Fig. 2A. The third peak, F23, exhib-
ited the highest calcium-binding capacity and was collected
for the subsequent purification (Fig. 2B).
Fraction 7, with a retention time of 21 min (Fig. 3A), pos-
a semi-preparative C18 RP-HPLC column (Fig. 3B). Fraction 7
from the preparative HPLC column was further fractionated
using analytical RP-HPLC, and the resultant fractions were
manually collected (Fig. 4A). Fraction 2 and fraction 4
were the active components obtained by analytic HPLC
(Fig. 4 B).
A 12
F2 0.5
Absorbance (214 nm)
F1 0.4
NaCl (mol/L)
8
0.3
4 F3 0.2
0.1
0 0.0
0 20 40 60 80 100 120 140
Fraction number
B
64
Calcium-binding capacity
(µg/mg peptide)
56
48
40
32
F1 F2 F3
Fig. 1 – (A) DEAE anion-exchange column chromatography
of whey protein hydrolysates. (B) Calcium-binding capacity
of F1, F2, and F3 separated from DEAE.
 journal of functional foods 10 (2014) 46–53 49

A mAU %
7 F24 A 4
1000 90
6
Absorbance (214 nm)

F22 2
F21 F23 80
5

Absorbance (214 nm)

Acetonitrile(%)
4 750 70
3 60
2 500 50
3 40
1
0 1 5 30
250
0 10 20 30 40 50 60 70 80 20
B Fraction number 10
66 0
Calcium-binding capacity

0.0 5.0 10.0 15.0 20.0 25.0 min

B
(µg/mg peptide)

63
90

Calcium-binding capacity
60

(µg/mg peptide)
80
57 70

54 60
F21 F22 F23 F24
50
1 2 3 4 5
Fig. 2 – (A) Sephadex G-25 gel filtration chromatography of
F2 derived from ion-exchange column chromatography. (B) Fig. 4 – (A) Reversed-phase high-performance liquid
The calcium-binding activities of fractions from G-25. chromatography (RP-HPLC) chromatography of fraction 7
derived from preparative HPLC. (B) Calcium-binding
capacity of fractions 1–5 from analytical RP-HPLC.

3.2. Identification of calcium-binding peptide by LC/MS

The molecular weight and amino acid sequence of fraction 2 higher than that of J. belengerii frame peptide (JFP) possessing
from HPLC analysis were determined using an LC/MS tandem the calcium-binding capacity of 70 μg/mg (Jung & Kim, 2007).
mass spectrometer. The fraction with a retention time of The general principle of metal ion coordination with ligands
5.84 min shown in Fig. 5(A) was identified to be Phe-Asp with follows acid–base theory. A hydrogen ion is dissociated from
a molecular weight of 280 Da (Fig. 5(B)). The result was con- the acid groups in ligands resulting in combination between
firmed by comparison with data from the National Center for the ligand and metal ion. Many metal-chelating peptides iso-
Biotechnology Information (NCBI) database. The calcium- lated from hydrolysates were reported, and most of them have
binding capacity of this peptide was determined to be 73.4 μg/ high content of Asp (Storcksdieck, Bonsmann, & Hurrell, 2007;
mg, binding equivalent content of calcium to casein-derived Swain, Tabatabai, & Reddy, 2002; Taylor, Martinez-Torres,
calcium phosphopeptide (CPP) with 76 μg/mg. Moreover, it was Romano, & Layrisse, 1986). Negatively charged COO− groups have
been reported to be potential binding sites, and the NH2 or NH
groups of amido bonds may also participate in coordination
of the chelation complexes (Wang, Li, Yang, Wang, & Shen,
A mAU
% 2009). Lv et al. (2009) have shown that the binding sites between
2000
90
soy protein hydrolysate and iron may be the carboxyl groups
80
Absorbance (214 nm)

of the Asp and Glu residues. Kim et al. (2007a) revealed that
Acetonitrile (%)

1500 2 70
8 9 10 an iron-binding peptide fractionated from heated whey hy-
13 60
7
1000 6 50 drolysate had higher percentage of Phe residues (16.58%). There-
3 40
1 45 fore, it appears that the isolated peptide containing Asp and
11 12 30
500 Phe may be of great benefit in generating high calcium affin-
20
10 ity. Furthermore, dipeptide or tripeptide was thought to promote
B
0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min metal ion absorption more effectively than high-molecular
Calcium-binding capacity

72 weight peptides in intestinal epithelial cells (Wang, Li, & Ao,

(µg/mg peptide)

2012).
66

60 3.3. Fluorescence spectra of FD and FD-Ca complex

54
1 2
Fig. 3 – (A) Preparative C18 reversed-phase high-
performance liquid chromatography (RP-HPLC) profile of
purified peptides. Thirteen fractions were collected for
determination of their calcium-binding activity. (B)
Calcium-binding activity of fractions 1–13 collected by
preparative RP-HPLC.
3 4 5 6 7 8 9 10 11 12 13 The aromatic amino acids phenylalanine, tyrosine, and tryp-
tophan can generate endogenous fluorescence at the appro-
priate excitation wavelength. The fluorescence absorption band
at 320 nm decreased as the calcium concentration increased.
In particular, the endogenous fluorescence decreased dramati-
cally when 5 μM CaCl2 was added to the FD solution (Fig. 6).
With the calcium ion concentration increased, the extent of
endogenous fluorescence extinction reduced. However, no re-
50 journal of functional foods 10 (2014) 46–53

5.84

8.0 5.23

6.0

AU
4.0 4.04
4.22
2.0
1.35
0.0 Time
-0.00 2.00 4.00 6.00 8.00 10.00 12.00

FD bMax
B D F yMax
100 280.97(M+H) +
120.00
a1
Intensity (%)

% 121.01
282.00
102.99
133.97
y1 282.98
13.01 93.01 235.99
199.98 217.00 363.83 381.05 391.20
0 M/z
50 100 150 200 250 300 350 400

Fig. 5 – ESI/MS spectrum of active fraction 2 from analytical HPLC. (A) Diode array of fraction 2 from Fig. 4. (B) Amino acid
sequence of the fraction with a retention time of 5.84 min deduced from the diode array and sequenced by LC/MS.

markable difference was observed when changing the con- contributed to the decrease in the fluorescence intensity (Uppal,
centration of CaCl2, which might be caused by overdose of Lakshmi, & Valentine, 2008).
calcium ion in the mixtures. In general, calcium ion may cause
fluorescence quenching of calcium-binding peptide, which likely
3.4. FTIR spectra of FD and FD-Ca complex

The characteristic FTIR absorption peak changes of the car-

200 FD boxylates and amides in FD could reflect the interaction of metal
FD +5 µM CaCl2 ions with organic ligand groups of the peptides. The two most
175 FD +10 µM CaCl2 important vibrational modes of amides are the amide-I vibra-
FD +15 µM CaCl2 tion and amide-II vibration, the amide-I vibration is primar-
150
FD +20 µM CaCl2 ily caused by stretching of C=O bonds, amide-II vibration is
Fluorescence intensity

125 assigned to deformation of N—H bonds and stretching of C—N

FD +25 µM CaCl2
bonds (Van der Ven et al., 2002; Zhou et al., 2012). The results
100 shown in Fig. 7 indicated that the wave numbers (1723 cm−1
and 1674 cm−1) of the amide-I band shifted to lower frequen-
75
cies (1679 cm−1 and 1580 cm−1) with addition of calcium. After
50 binding with calcium, the band at 1540 cm−1, corresponding to
the carboxyl group, shifted to 1417 cm−1 and became a deeper
25 valley. The absorption band at 1429 cm−1 reflected the contri-
bution of the N—H in the amide bond, upon coordination with
0
320 340 360 380 400 the calcium–peptide complex, the NH band shifted to 1307 cm−1.
Wavelength (nm) The peaks observed at 1192 cm−1 and 1143 cm−1 became shal-
lower and shifted to lower frequencies at 1307 cm −1 and
Fig. 6 – Fluorescence emission spectra of the peptide FD at 1209 cm−1 when FD combined with Ca2+ to form C—O–calcium.
various concentrations of Ca2+. In the range 500–800 cm−1, several absorption bands, which arose
 journal of functional foods 10 (2014) 46–53 51

shift to reveal the reaction between the calcium-binding peptide

and calcium ion. The low-field resonance signals, from 5.0 to
9.0 ppm, of the NMR spectrum (Fig. 8) were caused by the ali-
Relative transmissivity rate (%)

phatic group of phenylalanine, and the N—H bonds at 7.85 ppm

1429
shifted to a lower magnetic field of 8.19 ppm after FD binding

1540
3076 with calcium. In the high magnetic field area, the triple peaks

1143
1723
at 3.5–3.6 ppm corresponded to H spin coupling cracking in the

1192
α-H of phenylalanine or aspartic acid shifted to a lower mag-

1674
netic field of 4.04 ppm after chelation. The signals at 2.75 ppm

1135
and 2.91 ppm arose from the two β-H atoms of phenylala-

1307
1209
nine. With the addition of calcium, one peak shifted from
3419

2.75 ppm to 2.95 ppm and the other one shifted from 2.91 ppm

4000 3500 3000
Wavenumber (cm-1)
Fig. 7 – FTIR spectra of FD and the FD-Ca complex over the
wave number range from 4000 to 400 cm−1.
from vibration of the C—H and N—H bonds in the ion-binding
peptide (Chen et al., 2013), were not present in the peptide–
calcium complex spectra. Following coordination of the FD-
Ca complex, the N—Ca bond replaced the N—OH hydrogen
bonds and the absorption shifted from 3076 cm−1 to 3419 cm−1,
indicating that the principal binding sites of FD were the car-
boxyl and amino groups, as well as the peptide bonds.
3.5. High-resolution NMR spectroscopy of FD and
FD-Ca complex
The 1H NMR spectra reflect the distribution of the electron cloud
around a hydrogen nucleus through the changes of chemical
15801417
1679 FD to 3.19 ppm. Near the 2.5 ppm region, two β-Hs of aspartic acid
FD-Ca
shifted in opposite directions, one from 2.51 ppm to the low
magnetic field of 2.59 ppm, and the other from 2.44 ppm to
2500 2000 1500 1000 500
2.39 ppm. These changes in hydrogen atom spin coupling crack-
ing resonance signal peaks were caused by the chelation of FD
to calcium ion affecting the electron density around the FD
protons. When the electron density decreased, the shielding
effect consequently weakened and the resonance frequency
increased, the signal peaks therefore moved to lower mag-
netic fields.
3.6. Calcium-releasing percentage of FD-Ca complex
The calcium-releasing percentages of FD-Ca complex and CaCl2
at various pH values were shown in Fig. 9. Both FD-Ca and CaCl2
showed good calcium-releasing capacity, and the calcium-
releasing percentage reduced as pH increased. However,
calcium-releasing amount of FD-Ca decreased more slowly than
that of CaCl2. When pH was higher than 7.0, the releasing per-
centage of CaCl2 was significantly lower than that of FD-Ca
complex (Fig. 9). It is well known that the pH value of the human
intestinal tract is higher than 7.0, approximately pH 7.2,
FD-Ca complex would remain relatively high calcium-releasing

25000
2000 4000
Δ=0.48 3000 20000
Δ=0.34 1000
500 2000
1000 15000
0
0
10000
8.4 8.2 8.0 7.8 4.1 4.0 3.8 3.7 3.5 3.4
ppm ppm
5000

0
Δ=0.04
Δ=0.72
6000
Δ=0.05 -5000
4000
Δ=0. -10000
2000
-15000
0
3.2 3.0 2.8 2.6 2.4 -20000
ppm

12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2
ppm

Fig. 8 – 1H NMR spectral region from 0.5 to 10 ppm of FD and FD-Ca (the red one–FD; the green one–FD-Ca).
52 journal of functional foods 10 (2014) 46–53
100
95
Calcium-releasing ability (%)
90
85
80
75
2 3 4 5
pH
Fig. 9 – Calcium-releasing percentage of FD-Ca complexes
and CaCl2 at various pH values from 2.0 to 8.0.
percentage and keep dissolved in the basic environment of the
gastrointestinal tract, which could prevent calcium ion from
forming precipitate so that it could be effectively absorbed by
intestinal epithelial cells. The result suggests that the solubil-
ity of calcium supplements in the human gastrointestinal tract
is of great importance, and calcium nutritional supplements
with high solubility probably have prominent bioavailability
as reported by Wang, Zhou, Tong, & Mao, 2011.
4. Conclusion
In summary, a dipeptide FD with strong calcium-binding ca-
pacity was purified from whey protein hydrolysate, and the Asp
residue of Phe-Asp played an important role in binding to
calcium ion. FD-Ca chelate demonstrated both stability and
absorbability under either acidic or basic conditions. The
results suggest that whey protein possibly has the potential
to be used as the raw material to produce the calcium-
binding peptide as dietary supplements for osteoporosis in
functional foods.
Acknowledgments
This work was supported by the Fujian Natural Science Foun-
dation, China (No. 2013J01132) and the S&T projects of Fujian
Provincial Science & Technology Hall (Nos. 2012N0015 &
2012S0053).
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