2014 Volume 20 Issue 2 Pages 385-392
Carp egg phosphopeptide (EPP) with Ca binding activity was isolated from tryptic hydrolysate by ultrafiltration and hydroxylapatite chromatography. EPP mainly consists of 3.99 ± 0.12% P (w/w) and 20.36 ± 0.50% serine residues (w/w). In vitro EPP exhibited high Ca binding ability which was slightly lower than that of casein phosphopeptide (CPP) and could inhibit the formation of insoluble Ca salts. In vivo the effects of EPP on increasing Ca bioavailability were further studied in Ca-deficiency rats. All rats were randomly divided into five groups, one group served as the normal group to feed with a normal-Ca diet including CaCO3 for 8 weeks, another as the Ca-deficiency group to feed with a low-Ca diet for 8 weeks, the other three groups to feed with the low-Ca diet for 4 weeks and then randomly assigned to the control group and two experimental groups. The control group was switched to the normal-Ca diet for 4 weeks. The experimental groups were fed with the normal-Ca diet containing EPP and CPP for 4 weeks respectively (marked as the EPP group and CPP group). During the experimental period, Ca absorption and its accumulation in bone was significantly increased by EPP supplementation. The levels of serum Ca, bone mineral density, bone Ca content and biomechanical properties of the EPP group were significantly higher than those of control group (p < 0.05), but similar to the CPP group (p > 0.05). EPP is expected to become a novel Ca nutraceutical additive in food industry due to enhancing Ca bioavailability by its intake.
Ca is an important mineral element to maintain human health, which plays an important role in the formation of bones and teeth, neurotransmitter release, muscle composition, and heart beat regulation. Ninety-nine percent of Ca of human body exists in the bones, and the remaining 1% is in teeth, soft tissue, blood, and extracellular fluid (Straub, 2007). Although serum Ca level can be maintained in the normal range by bone resorption, dietary intake is the only source by which the body can replenish stores of Ca in bone (Choi et al., 2005). The absorption of Ca is influenced by many factors, such as chemical matrix of diet and nutritional, metabolic, and physiological status of the body (Pérez et al., 2008; Kerstetter, 1998). Ca deficiency can lead to many diseases such as osteoporosis and rickets variety (Nordin, 1997). Since Ca metabolism is a very complex process, the treatment of Ca deficiency is not simply to add Ca to diet. It is related to the absorption and utilization of Ca.
The early work of Mellander (1950) was the first indication that CPP owned the function to promote bone calcification of children suffering from rickets. Successively, other studies also observed enhancement of Ca absorption and bone Ca deposition in rats by oral intake of CPP (Mykkanen and Wasserman, 1980; Sato et al., 1986, 1991). Researchers believe that CPP has the function to bind metal ions and inhibit their precipitation due to containing large amounts of phosphorylated serine, which is just the key for CPP to promote body's Ca absorption (West, 1986; Holt et al., 1998). Ninety percent of Ca absorption occurs in the small intestine (Bronner, 2008). However, the weak alkaline condition in the small intestine makes Ca to be easily precipitated and be difficult to absorb. Studies have shown that CPP can maintain a higher concentration of soluble Ca in small intestine so as to promote Ca's absorption in the small intestine through passive diffusion absorption (Sato et al., 1986). Since phosphate groups endow CPP with high ability to promote Ca absorption, researchers devote themselves to develop other proteins or peptides rich in phosphate groups, such as phosvitin and its tryptic peptide, osteocalcin and fish bone peptides for Ca nutraceutical additive usage (Jiang and Ming, 2000; Jung et al., 2006; Jung and Kim, 2007; Nishmoto et al., 2003). However, some scholars have pointed out that the phosphorus-rich macromolecules such as phosvitin would resist the hydrolysis of protease (Mecham and Olcott, 1949; Goulas et al., 1996) and reduce Ca absorption (Ishikawa et al., 2007; Mineo et al., 2009), and we should obtain small molecules phosphorylated peptides through exogenous hydrolysis.
The global fishery production reached 178 million tons in 2011 (i), about 50% of which was discarded as inedible byproducts such as bone, skin, internal organs, head and eggs. Plentiful researches have been implemented on utilizing fishery byproducts (Chakraborty et al., 2011; Duan et al., 2009; Kongsri et al., 2013; Mori et al., 2013). However, the utilization studies on fish eggs, especially on freshwater fish eggs, are very scarce. Carp was one of the most production (over 3.8 million tons) freshwater fish species in the world (ii). In breeding season, the ovarian can weigh 10% to 25.81% of the overall weight. Many researches indicated that fish eggs also contain high phosphoprotein which is similar to hen phosvitin (Catherine et al., 2002; Inoue et al., 1971). This paper firstly researched the effect of EPP obtained from carp eggs through tryptic hydrolysis on promoting Ca bioavailability.
Preparation of carp egg phosphopeptide with Ca-binding activity Carp eggs obtained from industrial processing were provided by Meijia Co. (Shandong, China), and stored at −70°C before use. According to our previous research (Li and Huang, 2011), after defatted with 10 times volume of hexane/ethanol mixed solvent (3:1(V/V)) at 50°C for 6 h and dephosphorylated with 40 times volume of 0.1 mol/L NaOH for 2 h, carp eggs with proximate 30.5% dephosphorization degree then were digested with trypsin (enzyme/substrate: 3/100, substrate concentration: 2%, pH 8.0) at 49°C for 24 h. After incubation at 100°C for 5 min to inactivate the enzyme, the carp egg hydrolysate was centrifuged at 10,000 g for 20 min and filtered with 0.45 µm membrane. After demineralised on a Chelex 100 resin column (Bio-Rad, Richmond, CA, USA) the hydrolysate was divided into three fractions by ultrafiltration membrane system with 1 KDa and 5 KDa molecular weight cutoffs. The active fractions were concentrated and applied to a hydroxyapatite affinity column (16 × 200 mm, Macroprep ceramic HA type 1, Bio-Rad, Richmond, CA, USA) pre-equilibrated with 10 mM potassium phosphate buffer, pH 6.8. The Ca-binding fraction was divided into three fractions by step elution with 100 mM, 200 mM and 400 mM phosphate buffer (pH 6.8) at a flow rate of 3 mL/min and monitored at 220 nm. After demineralization the eluates were tested for Ca-binding activity. The fraction with the highest Ca affinity was pooled and lyophilized.
Chemical analysis Protein concentration in sample solutions was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. After demineralization the phosphorus content of sample solutions was determined according to the method of Chen et al. (1956). Amino acid analysis was conducted as follows. The samples were hydrolyzed with 6 mol/L HCl at 110°C for 24 h in vacuum-sealed ampoules. After neutralizing, evaporating and filtering with a glass filter, amino acid composition was determined with amino acid analyzer (Hitachi 835 - 50, Hitachi Co. Ltd., Japan).
In vitro Ca-binding assay Ca-binding assay was performed according to the previous method with the following modifications (Sato et al., 1991). Various concentrations of EPP up to 500 mg/L were mixed with 5 mM CaCl2 and 20 mM sodium phosphate buffer (pH 7.8). The mixture was stirred at 22°C for 30 min, and the pH was maintained at 7.8 with a pH meter. After filtered through 0.22 µm membranes to remove insoluble Ca phosphate salts, the Ca contents of the filtration were determined by a flame atomic absorption spectrometer. (AA-6300C, Shimadzu Co., Japan). Instrumental conditions were as follows: wavelength = 422.8 nm, slit = 0.8 nm, acetylene flow = 1.65 L/min, air flow = 14.0 L/min.
Experimental animals and diets Three-week-old male Sprague-Dawley rats were obtained from Lukang Pharmaceutical Co., Ltd. (Shandong, China). The rats were housed in individual stainless steel cages in a temperature- and humidity-controlled room (22 ± 2°C and 60 ± 5% relative humidity) with a 12 h light-dark cycle. The care of the rats in this study conformed to the Guidelines on the Use of Living Animals in Scientific Investigations (Biological Council, 1987). During the experimental period, rats were given free access to diets and deionised water. After fed for adaptability for one week, 50 rats were randomly divided into five groups , one group served as the normal group to feed with a normal-Ca diet including CaCO3 (17.5 g/kg) for 8 weeks, another as the Ca-deficiency group to feed with a low-Ca diet (0.175 g/kg CaCO3) for 8 weeks, the other three groups to feed with the low-Ca diet for 4 weeks and then randomly assigned to the control group and two experimental groups. The control group was switched to the normal-Ca diet for 4 weeks. The experimental groups were fed with the normal-Ca diet containing EPP (50 g/kg) and CPP (50 g/kg , Meiji Seika Co. Ltd., Tokyo, Japan) for 4 weeks respectively (marked as the EPP group and CPP group respectively). Body weights and amounts of food taken were recorded weekly. As shown in Table 1, all experimental diets were prepared according to the AIN-76 diet (Anonymous, 1977) with slight modification. Ca-free AIN-76 salt mix (Ralston Purina International Co., St Louis, MO, USA) was adopted in the present study and CaCO3 was added as the Ca source.
| Ingredients | Content (g/kg) | |||
|---|---|---|---|---|
| Low-Ca diet | Normal-Ca diet | |||
| Control | CPP | EPP | ||
| Casein | 200.0 | 200.0 | 150.0 | 150.0 |
| CPP | - | - | 50.0 | - |
| EPP | - | - | - | 50.0 |
| DL-Methionine | 3.0 | 3.0 | 3.0 | 3.0 |
| Corn starch | 499.8 | 482.5 | 482.5 | 482.5 |
| Sucrose | 150 | 150 | 150 | 150 |
| cellulose | 50.0 | 50.0 | 50.0 | 50.0 |
| Corn oil | 50.0 | 50.0 | 50.0 | 50.0 |
| Mineral mixa | 35.0 | 35.0 | 35.0 | 35.0 |
| CaCO3 | 0.175 | 17.5 | 17.5 | 17.5 |
| Vitamin mixb | 10.0 | 10.0 | 10.0 | 10.0 |
| Choline bitartrate | 2.0 | 2.0 | 2.0 | 2.0 |
CPP, casein phosphopeptide; EPP, carp egg phosphopeptide.
aCa-free AIN-76 mineral mix contains (g/kg): KH2PO4 500.00; NaCl 74.00; MgSO4 36.20; MgO 11.90; MnCO3·H2O 3.50; FeC6H5O7 6.00; ZnCO3·H2O 1.60; CuCO3 0.30; KIO3 0.01; Na2SeO3·H2O 0.01; CrK(SO4)2·12H2O 0.55; finely powdered sucrose 365.93.
bAIN-76A vitamin mixture contains (g/kg): thiamin HCl 0.60; riboflavin 0.60; pyridoxine HCl 0.70; niacin 3.0; Ca pantothenate 1.60; folic acid 0.20; biotin 0.02; vitamin B12 1.0; vitamin A palmitate 0.80; vitamin D3 0.25; vitamin E acetate 10.00; menadione sodium bisulfite 0.08; finely powdered sucrose, 981.15.
Ca balance study Ca balance evaluation was carried out for the last 4 days of the experiment. The amounts of food taken were recorded and the Urine and feces excreted were collected daily. Ca intake (Vi) could be determined by the recorded amounts of food taken. Urine and feces excreted were digested with HNO3 immediately after collection. Urinary Ca (Vu) and fecal Ca (Vf) were determined by a flame atomic absorption spectrometer. Ca absorption (Vab) was calculated as: Vab = Vi − Vf − Vu.
Serum and femur analysis After feeding experiment, all rats were fasted overnight and killed under pentobarbitone anesthesia. 4 mL blood collected from carotid bleeding was centrifuged (1000 g, 10 min, 4°C) to separate serum, and serum Ca, phosphor and alkaline phosphatase (ALP) activity was measured by an automatic analyser (ADVIA 2400, Siemens, USA). The right femurs were excised, cleared of muscles and connective tissues. The wet weight was recorded and the length was measured with vernier caliper. After the bone was heated at 580°C for 8 h, ash was dissolved in 10 mL of 6 mol/L HCl. The diluted femur solution was analysed for total Ca by flame atomic absorption spectrometry. Bone mineral density (BMD) of the distal region, defined as 5% of the whole length of the left femur, was determined by dual energy X-ray absorptiometry (Lunar Corp., USA). The left femurs were applied to an electronic universal testing instrument (Z005, Zwick Co., Germany) to conduct three-point-bending biomechanical test and measure the maximal load, elastic load and stiffness. The span was 20 mm and loading speed was 1 mm/min.
Statistical analysis Data were presented as means ± standard deviations of triplicate determinations. A least significant difference (LSD) test was performed using the SPSS software program (SPSS Inc., Chicago, IL, USA) to evaluate the mean differences between the measurements at the 5% confidence level.
Chemical analysis of EPP After ultrafiltration classification, the fraction (marked as Fu) with MW 1 – 5 KDa was obtained from carp egg tryptic hydrolysate (marked as Hy) and showed the best activity to bind calcium. Many studies have shown that biologically active peptides usually contain 3 – 20 amino acid residues and the molecular weight of discovered peptides with calcium binding activity are mostly from 1 KDa to 3.5 KDa (Jung et al., 2005, 2006, 2007; Jiang and Mine, 2000; Lee and Song, 2009). Fu was then isolated by hydroxyapatite chromatography and the fraction (marked as EPP) eluted with 400 mM phosphate buffer showed the highest calcium binding activity. The P, protein and serine contents of Hy, Fu and EPP were shown in Table 2. One notable feature was the high concentration of P and serine residues. EPP mainly consists of 3.99 ± 0.36% P (w/w) and 20.36 ± 0.50% serine (residues/100 residues). The results of an alkali-catalyzed β-elimination reaction suggested that all the phosphorus in the phosphoprotein is present in the form of monoesters linked to the hydroxyl groups of serine. The ratio of serine residue and P (mol/mol) decreasing from 2.10 to 1.52 indicated that more than 60% serine residue was phosphoserine after hydroxyapatite chromatography. One kind of phosphorus protein was obtained from Pacific Herring eggs, whose phosphorus content is up to 10.6% and serine residue accounts for 2/3 of all number of amino acids (Inoue, 1971).
| Sample | P (%, w/w) | protein (%, w/w) | serine residues (residues/100 residues) | Ratio of serine residue and P (mol/mol) |
|---|---|---|---|---|
| Hy | 0.68 ± 0.03 | 91.2 ± 0.3 | 6.81 ± 0.3 | 3.16 |
| Fu | 1.37 ± 0.04 | 89.4 ± 0.3 | 9.23 ± 0.3 | 2.10 |
| EPP | 3.99 ± 0.12 | 85.7 ± 0.5 | 20.36 ± 0.5 | 1.52 |
Hy, carp egg tryptic hydrolysate; Fu, the fraction after ultrafiltration with molecular weight 1 – 5KDa.
Ca binding activity of EPP in vitro Hydroxyapatite chromatography was very effective to separate peptide with calcium-binding activity due to its high affinity to the peptide. By hydroxyapatite chromatography, the peptides and proteins with calcium binding activity have been successfully separated and obtained from fish bones and bullfrog bone (Jung et al., 2005, 2006, 2007; Dohi, 1987). The results showed that after hydroxyapatite chromatography, the calcium binding activity of EPP was significantly increased and slightly lower than CPP (p < 0.05) (Figure 1). 200 mg/L EPP could bind 25.1 ± 0.5 mg/L Ca in the supernatant after the formation of insoluble salts, while in the treatment of 200 mg/L CPP, 28.4 ± 0.4 mg/L soluble Ca was analysed.
Ca-binding activity of EPP in vitro.
Hy, carp egg tryptic hydrolysate; Fu, the fraction after ultrafiltration with molecular weight 1 – 5 KDa; EPP, carp egg phosphopeptide; CPP, casein phosphopeptide. Various concentrations of protein up to 500 mg/L were mixed with 5 mM CaCl2 and 20 mM sodium phosphate buffer (pH 7.8). The mixture was stirred at 22°C for 30 min, and the pH was maintained at 7.8 with a pH meter. After removal of insoluble Ca phosphate salts and filtration with 0.22 µm membranes, Ca contents of the supernatant were determined by a flame atomic absorption spectrometer. The experiments were performed in triplicate. Values are means, with standard deviations represented by vertical bars.
There was a positive correlation between the phosphorus content of peptide and its Ca binding activity. The content of phosphorus may be critical to calcium binding ability of the phosphorus peptides. It was verified that CPP could prevent the formation of insoluble calcium in the small intestine and improve calcium absorption (Sato et al., 1986, 1991). The cause may lie in the fact that it possesses the structure with phosphoserine cluster (West, 1986; Holt et al., 1998).
The solubility of Ca is dependent on the concentration of EPP, which indicates that the binding of peptide and calcium does not comply with the stoichiometry. For example, 200 mg/L EPP contains 0.26 mmol P and would bind 0.13 ± 0.01 mmol Ca. However 200 mg/L EPP bound 25.1 ± 0.5 mg Ca, namely, 0.63 ± 0.01 mmol Ca, which means there is considerable inorganic phosphate present but that is not precipitated. This observation is consistent with the idea of the formation of small calcium phosphate nanoclusters stabilized by phospho-serine peptides (Zong et al., 2012; Holt et al., 1996).
Effects of EPP on body weight and Ca absorption of rats In the Ca-deficiency group, rats became inactive, and their hair lacked luster and became coarse. The body weight of this group was significantly lower than the rest of the groups (Table 3). All three Ca supplement methods can significantly increase the weight gain rate in rats and improve the malnutrition condition due to early Ca deficiency. Although there was no significant difference in the body weight among the control group, EPP group and CPP group, yet the body weight of the EPP group and the CPP group was closer to the normal group, while that of the control group was significantly lower than the normal group.
| Group | Body weight (g) | Vi (mg/d) | Vf (mg/d) | Vu (mg/d) | Vac (mg/d) |
|---|---|---|---|---|---|
| Normal | 355.67 ± 21.18a | 96.21 ± 15.63a | 45.47 ± 4.21a | 1.72 ± 0.33a | 49.02 ± 6.27b |
| Ca-deficiency | 216.24 ± 29.87c | 10.87 ± 1.68b | 1.58 ± 0.23c | 0.71 ± 0.03b | 8.58 ± 1.13c |
| Control | 311.33 ± 20.69b | 98.32 ± 7.51a | 41.57 ± 2.15a | 1.53 ± 0.21a | 55.22 ± 4.22b |
| EPP | 342.52 ± 28.61ab | 112.24 ± 9.23a | 35.71 ± 2.14b | 1.32 ± 0.19a | 75.21 ± 6.36a |
| CPP | 347.11 ± 24.36ab | 98.96 ± 8.33a | 29.99 ± 4.18b | 1.40 ± 0.12a | 67.57 ± 5.22a |
a–c Any means followed by different superscript letters are significant different (p < 0.05)
Except the Ca-deficiency group controlled at a low level of Ca intake, the Ca intake and the urine Ca in all other groups had no significant difference. The fecal Ca of the EPP group and the CPP group was significantly lower than the control group and the normal group, while the Ca absorption was significantly higher than these two groups. The function of the EPP and the CPP in promoting rats' weight gain, reducing fecal Ca, and increasing the Ca absorption was no significant differences. The body's Ca absorption rate is generally 30% – 80%. When the intake of Ca is insufficient, Ca is mainly absorbed in the near-end of the small intestine by active transport means, while Ca intake is sufficient, Ca is mainly absorbed in the distal of the small intestine by passive diffusion means (Hoenderop et al., 2005). However, the alkaline conditions in the distal of the small intestine will lead Ca to precipitation and inhibit its absorption. In this experiment, the Ca absorption of the EPP group was significantly higher than the control group, indicating EPP may also inhibit the precipitation of Ca in the distal of small intestine and promote the absorption of Ca by formation of small calcium phosphate nanoclusters. As reported by Gao et al. (2008), nanometer pearl powder could increase Ca absorption and bioavailability.
Meanwhile, we also observed that the absorption rate of the EPP group and the CPP group exceeded the normal group, indicating that Ca-deficiency rats in the early state will activate the active means of transport so as to make the Ca absorption rate higher than the normal level and improve their bodies' Ca deficiency physiological conditions.
Effects of EPP on Serum Calcium, phosphor and ALP of rats When the Ca in body is sufficient, the intestinal absorption of Ca will deposit in the bones through blood, promote bone to grow, increase bone density and bone strength, and make the serum Ca remain at normal levels (Bronner, 2008). When the body is in lack of Ca, serum Ca will decrease. At this time, the parathyroid hormone (PTH) will mobilize Ca of bone into blood to maintain blood Ca's stability. At the mean time, the bone osteoblasts will be extremely active. They will synthesize a large quantity of ALP and released into the blood (iii). Table 4 shows that the serum Ca in the Ca-deficiency group was significantly lower than the other four groups, while the ALP level was significantly higher than the other four groups (p < 0.05). However, Addition EPP to the normal diet could significantly improve the condition of low serum Ca symptom and made the ALP level remain stable. The serum P was not observed significant differences in all groups.
| Group | Serum Ca (mmol/L) | Serum P (mmol/L) | ALP (U/L) |
|---|---|---|---|
| Normal | 2.51 ± 0.18b | 2.17 ± 0.11a | 181.33 ± 21.42b |
| Ca-deficiency | 1.82 ± 0.17c | 1.99 ± 0.34a | 244.24 ± 27.53a |
| Control | 2.39 ± 0.14b | 2.11 ± 0.12a | 186.88 ± 22.94b |
| EPP | 2.88 ± 0.09a | 2.24 ± 0.27a | 175.21 ± 31.21b |
| CPP | 2.91 ± 0.12a | 2.35 ± 0.37a | 179.52 ± 19.36b |
a–c Any means followed by different superscript letters are significant different (p < 0.05)
Effects of EPP on bone growing of rats To evaluate the effects of EPP on bone Ca deposition, we tested femur length, weight, bone Ca content, and bone mineral density (Table 5). There were no significant differences in bone length of each group. For bone weight, there were no significant differences among the three kinds of Ca supplement group, whose bone weight levels could reach that of the normal group. However, in terms of bone density and Ca content, the EPP group and the CPP group was significantly higher than the control group, but similar to the normal group.
| Group | Femurs Length (mm) | Femurs Weight (g) | Bone Mineral Density (g/cm2) | Bone Ca Content (mg) |
|---|---|---|---|---|
| Normal | 33.8 ± 1.3a | 1.23 ± 0.05a | 0.185 ± 0.018a | 140.43 ± 6.21a |
| Ca-deficiency | 32.1 ± 1.8a | 0.74 ± 0.06b | 0.093 ± 0.012c | 67.86 ± 8.79c |
| Control | 33.3 ± 1.6a | 1.09 ± 0.12a | 0.13 ± 0.15b | 118.33 ± 4.32b |
| EPP | 34.0 ± 1.1a | 1.21 ± 0.09a | 0.183 ± 0.021a | 138.57 ± 7.11a |
| CPP | 34.0 ± 1.3a | 1.22 ± 0.14a | 0.186 ± 0.023a | 143.86 ± 7.73a |
a–c Any means followed by different superscript letters are significant different (p < 0.05)
The growth and development of bone is a constant conversion process during bone formation and bone resorption. During the early period of the formation of bone mass peak, the bone formation is higher than the bone resorption (Nordin, 1997). Thus, the bone mass accumulates and increases until bone mass peak term (35 – 40 years old). When the body is in Ca deficiency state, the bone lacks the mineralized Ca, resulting in the decrease of bone mineral density (Kenny and McCoy, 1997). Moreover, the bone Ca will be released to the blood to maintain blood Ca balance, which will intensify bone density to decrease and cause bone Ca lost (Kaastad et al., 1997). Tsuchita et al. (1996) reported that CPP-Ca could effectively inhibit senile bone loss in ovariectomized rats. In present experiment, EPP also could significantly increase bone mass, bone Ca content and bone mineral density.
Effects of EPP on Femur biomechanical indexes of rats To further confirm whether EPP could promote the effects of rats' bone Ca deposition, we conducted a rat femur biomechanical test. Biomechanics is the science which researches the biological effects of rats' bone under loads, the purpose of which is to show the ability of anti-fracture of bone acted upon by an external force. Turner and Burr (1993) reported that the ability of anti-fracture of femur was proportional to the square of bone mineral density. In the Ca-deficiency growing rats, abnormal spatial bone structure was observed by the histomorphometry method (Hong et al., 2002). The present test results (Table 6) showed that although the three kinds of Ca supplement methods can significantly increase the maximum load, elastic load and stiffness of the Ca-deficiency rats' femurs, yet all parameters of the EPP group and the CPP group were significantly higher than the control group. Moreover, their elastic load and stiffness were significantly higher than the normal group.
| Group | Maximum Load (N) | Elastic Load (N) | stiffness (N/mm) |
|---|---|---|---|
| Normal | 187.5 ± 15.6ab | 148.0 ± 10.4b | 169.4 ± 11.3ab |
| Ca-deficiency | 153.5 ± 18.8c | 106.7 ± 6.5c | 115.9 ± 8.8c |
| Control | 174.0 ± 13.5b | 141.7 ± 21.7b | 156.0 ± 5.1b |
| EPP | 199.9 ± 12.7a | 170.4 ± 6.2a | 180.6 ± 12.8a |
| CPP | 200.5 ± 23.0a | 169.7 ± 6.9a | 180.1 ± 12.5a |
a–c Any means followed by different superscript letters are significant different (p < 0.05)
CPP was obtained from whole milk cheese by hydrolyzed with trypsin and contained a series of fragment: αs1-CN (43 – 58), 2P; αs1-CN (59 – 79), 5P; αs2-CN (46 – 70), 4P; β-CN (1 – 28), 4P; β-CN (2 – 28), 4P; and β-CN (33 – 48), 1P. EPP isolated from carp egg tryptic hydrolysate was also abundant in phosphoserine and could inhibit Ca precipitation by formation of small calcium phosphate nanoclusters which is stabilized by phosphoserine. In the present study, we proved that the beneficial effects of EPP on inhibiting the formation of insoluble Ca salts in vitro and enhancing Ca bioavailability in vivo were similar to those of CPP. Furthermore, EPP was derived from fish eggs which were usually discarded as inedible byproducts. Thus, EPP is expected to become a novel Ca nutraceutical additive in food industry.
Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 31101379).
