A specific peptide with calcium chelating capacity isolated from whey protein hydrolysate

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 b

College of Bioscience and Biotechnology, Fuzhou University, Fuzhou 350002, China 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 PheAsp has the potential to be used as a calcium-binding ingredient in dietary supplements. © 2014 Elsevier Ltd. All rights reserved.

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 proteins whose solubility decreases in acid or heat conditions. The modification of whey protein has become an immediate

☆

Co-first authors. * Corresponding author. Tel.: +86 591 22866375; fax: +86 591 22866278. E-mail address: [email protected] (S.Y. Wang). http://dx.doi.org/10.1016/j.jff.2014.05.013 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

area of research focus all over the world. Enzymatic modification 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 investigated 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

journal of functional foods 10 (2014) 46–53

could increase the bone density in children and it is essential among the middle-aged and the aged to prevent osteoporosis (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 supplements 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 environment (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, obtained from proteolytic digestion, have shown the considerable 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 utilizing the hydrolyzed peptides from whey protein as calciumbinding 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.

equilibrating buffer and 20 mM Tris-HCl buffer (pH 9.0). Afterwards, 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 determined. The peak exhibiting the strongest ability was collected 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 purified 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 preparation for RP-HPLC. Runs were conducted with a liner gradient 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.

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 hydrolyzed using Flavourzyme and Protamex (2:1, w/w) with a substrate:enzyme ratio of 25:1 (w/w) at 49 °C for 7 h. Hydrolysate 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 hydrolysate (WPH), was lyophilized and stored at −20 °C for subsequent purification.

2.3.

47

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

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 solution 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 competitively combining with CaCl2. The reaction mixture was centrifuged 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 colorimetric 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 expressed as mean ± standard deviation (SD).

2.6. Structural characterization of peptide–calcium complex Preparation of peptide–calcium complex

Fluorescence spectra

Fluorescence spectra were measured to monitor conformational 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 emission 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 recorded 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 software (Thermo Nicolet Co.; Madison, WI, USA).

2.6.4.

1

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 deuterium 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 refrigerated 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 colorimetric 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.

Results and discussion

3.1.

Purification of calcium-chelating peptide

The eluted fractions separated from DEAE anion exchange chromatography 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 condition 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 filtration chromatograph column, size-dependent fractionation of the samples was shown in Fig. 2A. The third peak, F23, exhibited 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), possessing the highest calcium-binding capacity, was isolated on 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

0.4

8

0.3 0.2

F3

4

0.1 0 0

B

0.5

F2 F1

20

40

60

80

100

120

140

NaCl (mol/L)

2.6.2.

3.

Absorbance (214 nm)

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.

Calcium-binding capacity (µg/mg peptide)

2.6.1.

Chicago, IL, USA). Data were presented as mean ± standard deviation (SD). Statistical significance was determined by oneway analysis of variance (ANOVA) followed by Duncan’s multiple range test. Statistical analysis was performed using the software Origin 8.0 (Origin Lab Co.; USA).

0.0

Fraction number

64 56 48 40 32 F1

F2 F3

Statistical analysis

All experiments were determined in triplicate. Statistical analysis was performed using the SPSS software program (SPSS Inc.;

Fig. 1 – (A) DEAE anion-exchange column chromatography of whey protein hydrolysates. (B) Calcium-binding capacity of F1, F2, and F3 separated from DEAE.

49

journal of functional foods 10 (2014) 46–53

mAU

0 B

10

20

30

F23

40

50

60

70

80

Fraction number

66

Calcium-binding capacity (µg/mg peptide)

F22

F21

A

750 500

3 5

1

250

5.0

10.0

15.0

20.0

25.0

min

B

60 57 54 F21

F22 F23

F24

Fig. 2 – (A) Sephadex G-25 gel filtration chromatography of F2 derived from ion-exchange column chromatography. (B) The calcium-binding activities of fractions from G-25.

90 80 70 60 50 1

2

3

4

5

Fig. 4 – (A) Reversed-phase high-performance liquid chromatography (RP-HPLC) chromatography of fraction 7 derived from preparative HPLC. (B) Calcium-binding capacity of fractions 1–5 from analytical RP-HPLC.

Identification of calcium-binding peptide by LC/MS

The molecular weight and amino acid sequence of fraction 2 from HPLC analysis were determined using an LC/MS tandem mass spectrometer. The fraction with a retention time of 5.84 min shown in Fig. 5(A) was identified to be Phe-Asp with a molecular weight of 280 Da (Fig. 5(B)). The result was confirmed by comparison with data from the National Center for Biotechnology Information (NCBI) database. The calciumbinding capacity of this peptide was determined to be 73.4 μg/ mg, binding equivalent content of calcium to casein-derived calcium phosphopeptide (CPP) with 76 μg/mg. Moreover, it was

mAU

%

2000

90 80

2

1500

8 6

1000

1

3

70

9 10

13

7

60 50 40

45 11

500

30

12

20 10

Calcium-binding capacity (µg/mg peptide)

B

0 0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

min

72

Acetonitrile (%)

A

Absorbance (214 nm)

90 80 70 60 50 40 30 20 10

2

0 0.0

63

3.2.

%

4

1000

Acetonitrile(%)

F24

Absorbance (214 nm)

Absorbance (214 nm)

7 6 5 4 3 2 1 0

Calcium-binding capacity (µg/mg peptide)

A

higher than that of J. belengerii frame peptide (JFP) possessing the calcium-binding capacity of 70 μg/mg (Jung & Kim, 2007). The general principle of metal ion coordination with ligands follows acid–base theory. A hydrogen ion is dissociated from the acid groups in ligands resulting in combination between the ligand and metal ion. Many metal-chelating peptides isolated from hydrolysates were reported, and most of them have high content of Asp (Storcksdieck, Bonsmann, & Hurrell, 2007; Swain, Tabatabai, & Reddy, 2002; Taylor, Martinez-Torres, 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, 2009). Lv et al. (2009) have shown that the binding sites between soy protein hydrolysate and iron may be the carboxyl groups of the Asp and Glu residues. Kim et al. (2007a) revealed that an iron-binding peptide fractionated from heated whey hydrolysate had higher percentage of Phe residues (16.58%). Therefore, it appears that the isolated peptide containing Asp and Phe may be of great benefit in generating high calcium affinity. Furthermore, dipeptide or tripeptide was thought to promote metal ion absorption more effectively than high-molecular weight peptides in intestinal epithelial cells (Wang, Li, & Ao, 2012).

66

3.3.

60 54 1

2

3

4

5

6

7

8

9

10 11 12 13

Fig. 3 – (A) Preparative C18 reversed-phase highperformance 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.

Fluorescence spectra of FD and FD-Ca complex

The aromatic amino acids phenylalanine, tyrosine, and tryptophan can generate endogenous fluorescence at the appropriate excitation wavelength. The fluorescence absorption band at 320 nm decreased as the calcium concentration increased. In particular, the endogenous fluorescence decreased dramatically 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

A

AU

8.0

5.23

6.0 4.04

4.0

4.22

2.0 1.35

0.0 -0.00

2.00

4.00

FD

D

B 100

Intensity (%)

6.00

10.00

Time 12.00

bMax yMax 280.97(M+H) +

F

120.00 a1 F

%

8.00

121.01 282.00

102.99

133.97 y1 235.99 199.98 217.00

93.01

13.01 0 50

100

150

200

250

282.98

363.83 381.05 391.20 M/z 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 concentration of CaCl2, which might be caused by overdose of calcium ion in the mixtures. In general, calcium ion may cause fluorescence quenching of calcium-binding peptide, which likely

FD FD +5 µM CaCl2

200 175

FD +10 µM CaCl2 FD +15 µM CaCl2

Fluorescence intensity

150

FD +20 µM CaCl2

125

FD +25 µM CaCl2

100 75 50 25 0 320

340

360

380

400

Wavelength (nm)

Fig. 6 – Fluorescence emission spectra of the peptide FD at various concentrations of Ca2+.

contributed to the decrease in the fluorescence intensity (Uppal, Lakshmi, & Valentine, 2008).

3.4.

FTIR spectra of FD and FD-Ca complex

The characteristic FTIR absorption peak changes of the carboxylates and amides in FD could reflect the interaction of metal ions with organic ligand groups of the peptides. The two most important vibrational modes of amides are the amide-I vibration and amide-II vibration, the amide-I vibration is primarily caused by stretching of C=O bonds, amide-II vibration is assigned to deformation of N—H bonds and stretching of C—N bonds (Van der Ven et al., 2002; Zhou et al., 2012). The results shown in Fig. 7 indicated that the wave numbers (1723 cm−1 and 1674 cm−1) of the amide-I band shifted to lower frequencies (1679 cm−1 and 1580 cm−1) with addition of calcium. After binding with calcium, the band at 1540 cm−1, corresponding to the carboxyl group, shifted to 1417 cm−1 and became a deeper valley. The absorption band at 1429 cm−1 reflected the contribution of the N—H in the amide bond, upon coordination with the calcium–peptide complex, the NH band shifted to 1307 cm−1. The peaks observed at 1192 cm−1 and 1143 cm−1 became shallower and shifted to lower frequencies at 1307 cm −1 and 1209 cm−1 when FD combined with Ca2+ to form C—O–calcium. In the range 500–800 cm−1, several absorption bands, which arose

51

1540 1429

4000

3500

15801417

1679

3419

1307 1209 1135

1192 1143

1674

1723

3076

Relative transmissivity rate (%)

journal of functional foods 10 (2014) 46–53

3000

2500

2000

FD FD-Ca

1500

1000

500

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 FDCa 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 carboxyl 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

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 aliphatic group of phenylalanine, and the N—H bonds at 7.85 ppm shifted to a lower magnetic field of 8.19 ppm after FD binding with calcium. In the high magnetic field area, the triple peaks at 3.5–3.6 ppm corresponded to H spin coupling cracking in the α-H of phenylalanine or aspartic acid shifted to a lower magnetic field of 4.04 ppm after chelation. The signals at 2.75 ppm and 2.91 ppm arose from the two β-H atoms of phenylalanine. With the addition of calcium, one peak shifted from 2.75 ppm to 2.95 ppm and the other one shifted from 2.91 ppm to 3.19 ppm. Near the 2.5 ppm region, two β-Hs of aspartic acid 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 2.39 ppm. These changes in hydrogen atom spin coupling cracking 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 magnetic 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 calciumreleasing 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 percentage 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 4000

2000 Δ=0.48

1000

Δ=0.34

20000

3000 2000

500

15000

1000 0 8.4

8.2

8.0 ppm

0 4.1

7.8

10000

4.0 3.8 3.7 3.5 3.4 ppm

5000 0

Δ=0.04

Δ=0.72

6000

Δ=0.05

-5000

4000

-10000

Δ=0.

2000 -15000 0

12

11

10

9

8

7

6

5

3.2

3.0

4

3

2.8 2.6 ppm

2

1

2.4

-20000

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).

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journal of functional foods 10 (2014) 46–53

100

CaCl2 FD-Ca

Calcium-releasing ability (%)

95

90

85

80

75 2

3

4

5 pH

6

7

8

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 solubility 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 capacity 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 calciumbinding peptide as dietary supplements for osteoporosis in functional foods.

Acknowledgments This work was supported by the Fujian Natural Science Foundation, China (No. 2013J01132) and the S&T projects of Fujian Provincial Science & Technology Hall (Nos. 2012N0015 & 2012S0053).

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