Original papers
Preparation and Characterization for Peptide-Chelated Calcium of Deer Bone
2018 Volume 24 Issue 4 Pages 717-728
Details
2018 Volume 24 Issue 4 Pages 717-728
The deer bone peptides (DBP) was used to chelate with Ca2+ to form the deer bone peptides chelated calcium (DBPCC), which could be a calcium nutraceutical supplement. The optimal condition for the chelation reaction was that 1.535 mg mL−1 DBP reacted with 7.5 mmol L−1 CaCl2 at pH 7.0 and 40 °C for 15 min. Negatively charged amino acids, positively charged amino acids and hydrophobic amino acids in the DBPCC were probably important to the chelation reaction. And the DBP could chelate with Ca2+ through metal-binding sites (carboxyl oxygen atoms and amino nitrogen atoms). The Ca2+ would cause the DBP folding and conformation modification. Furthermore, the DBPCC was stable under ileum conditions (pH 7.0, 37 °C), indicating that it would be well absorbed in the human body and had the potential to improve the traditional calcium nutrition fortifier.
Calcium is an essential macronutrient in the human body. The adequate dietary intake of calcium can reduce the risk of osteoporosis (Cashman, 2007), hypertension (Osborne et al., 1996) and colon cancer (Lipkin et al., 1993). The effect of calcium absorption is dependent upon the amount of soluble calcium absorbed in the intestines of the human body with the existence of calcium binding protein (CaBP), where calcium is still soluble in the alkali gastrointestinal environment (Fordtran and Locklear, 1966, Gleason et al., 1979, Lin et al., 2015). However, owing to lactose intolerance and dietary habits, the Asian intake less calcium from food such as milk and cheese products, resulting in calcium deficiency and causing health problems. Therefore, calcium is usually fortified as calcium salts, but the substances such as phosphate and oxalate will react with calcium salts to lower the bioavailability (Chen et al., 2014). Calcium carbonate and calcium citrate are now commonly used as calcium supplements in clinical practice (Straub, 2007). Nevertheless, calcium carbonate should be taken with meals to optimize absorption, and calcium citrate is recommended in patients with achlorhydria or who are taking histamine-2 blockers or protein-pump inhibitors (Straub 2007). As a result, both of calcium carbonate and calcium citrate are not prevailing in human daily diets.
The test of calcium absorption and bone properties in vivo showed that the calcium supplement efficiency of the peptide-chelated calcium was better than that of the calcium carbonate (Guo et al., 2015, Chen et al., 2014). Until now, it has been demonstrated that the peptide-chelated calcium might be a typical calcium fortifier to improve bone health of human body. For example, several investigators had suggested that the peptides from whey protein (Xixi et al., 2015), fish scales (Guo et al., 2015, Chen et al., 2014) and soybean (Bao et al., 2008, Lv et al., 2013b) could chelate with Ca2+ and the reaction products were stable in the gastrointestinal tract to promote calcium absorption in vivo. Actually, the deer bone is rich in collagen which can be hydrolyzed into peptides, but there were little studies about the peptides from deer bone chelated with Ca2+ as calcium fortifier. Traditionally, the deer bone has been regarded as Chinese medicine because of the high nutritional value and pharmacological activity. Researchers had also found that deer bone extract could be a potent nutraceutical agent for osteoarthritis treatment (Lee et al., 2014a, Lee et al., 2014b). It had been proved that deer bone polypeptides could improve pathological changes in the bone microarchitecture of osteoporosis rats (Li-ping et al., 2016). Besides, deer bone extract might also be a useful functional agent for the prevention against neurodegenerative disorders involving oxidative stress (Kim et al., 2014). So far deer bone is mainly used to make bone glue, animal feed, or handicrafts, not for chelating agent to form the deer bone peptides chelated calcium (DBPCC).
The objective of this study was to prepare deer bone peptides (DBP) from hydrolyzing deer bone powder, and then chelate the peptides with calcium to form the DBPCC. Afterwards, the chemical components, structure, molecular weight distribution, size distribution and amino acid components of the DBP and the DBPCC were investigated to explore the binding sites to analyze the chelating mechanism about the peptide-calcium chelation. It might advance the development of calcium supplementation and provide basic theories for the utilization of the deer bone.
2.1 Dear bone and reagents Fresh deer bone was purchased (15th Mar., 2017) from Lishi Deer Farm in Changchun, Jilin Province and stored at 4 °C until usage. Protamex (1.5 AU-N g−1) was obtained from Novozymes Groups (Copenhagen, Denmark). Calcium chloride was bought from Beijing Chemical Industry Group Co., Ltd (Beijing, China). All of the other chemical reagents were analytical grade.
2.2 The preparation of deer bone powder and hydrolysis of the deer bone protein The deer leg bone was crushed into bone block size (10±5 cm). Then remove the tendons, skin, bone marrow, and a small amount of meat on the deer bone blocks. Cleaned the bone blocks with distilled water and drained them, and then cooked them for 1h in an autoclave (MY-QC60A5, Guangdong Midea Electric Manufacturing Co., Ltd, China). Removed the cooking liquid, rinsed the deer bone blocks with cold water to wipe off the grease, dried the deer bone for 8 hours at 60 °C and then further crushed the deer bone blocks into deer bone powder using a crusher (FW77, Tianjin Taisite Instrument Co., Ltd, China). The mixture of deer bone powder and deionized water (5 % substrate concentration) was adjusted to pH 6.5 with HCl (1 mol L−1) and then hydrolyzed by protamex using an enzyme/substrate ratio of 8/1000 (w/w) at 55 °C for 4h. After inactivated the enzyme for 10 min at 100 °C, the deer bone hydrolysate was centrifuged at 3000 rpm for 20 min at room temperature and the supernatant was then lyophilized as the DBP and stored at −20 °C for further analysis.
2.3 Optimization for the reaction condition of preparing the DBPCC The factors including the DBP concentration, the concentration of Ca2+, pH value, the temperature, and the reaction time to the reaction system were investigated to determine the appropriate chelation reaction parameters based on the chelating rate and the quantity of peptide chelated calcium (Table 1). Initially, the DBP powder was dissolved in distilled water and mixed with calcium chloride solution (0.1 mol L−1) until the concentration of the DBP and Ca2+ in this system was 1.535 mg mL−1 and 7.5 mmol L−1, respectively. The pH value of the reaction mixtures was adjusted to pH 7.0 by the addition of NaOH (0.05 mol L−1) or HCl (0.05 mol L−1), and the chelation reaction was incubated at 40 °C for 30 min with continuous stirring in water bath shaker. The obtained composite was lyophilized and then washed by absolute alcohol for three times to remove the free amino acids and Ca2+ by centrifuging at 3000 rpm for 30 min. The precipitates were collected and lyophilized as the DBPCC for the property analysis.
| Calcium concentration (mmol L−1) | DBP concentration (mg mL−1) | Time (min) | Temperature (°C) | pH value |
|---|---|---|---|---|
| 1.0 | 0.1535 | 5 | 30 | 3.0 |
| 2.5 | 0.7675 | 15 | 40 | 5.0 |
| 5.0 | 1.535 | 30 | 50 | 7.0 |
| 7.5 | 2.3025 | 45 | 60 | 9.0 |
| 10.0 | 3.07 | 60 | 70 | 11.0 |
2.4 Determination of the ability of peptides chelated calcium After the DBP enzymatic hydrolysates reacted with calcium chloride at the predetermined conditions, the reaction solution was diluted 50 times. The KCl was added in the solution with a final concentration of 0.5 M KCl as the ionic strength regulator. Then the free calcium content in the solution was measured with a Ca2+ selective electrode. And then two indicators of the quantity of peptide chelated calcium (Eq. 1) and the chelating rate (Eq. 2) were evaluated for the ability of peptide chelated calcium.
 |
Where Cb is the quantity of peptide chelated calcium (mg g−1), ωt is the quantity of total calcium, ωf is the quantity of free calcium, and ωpt is the quantity of protein.
 |
Where Cr is the chelating rate (%), ωt is the quantity of total calcium, and ωf is the quantity of free calcium.
2.5 Chemical analysis Protein, lipid, moisture and ash of the deer bone, the DBP and the DBPCC were determined according to the Association of Official Analytical Chemists method (AOAC, 1995). Crude protein was estimated from the total nitrogen multiplied by 6.25.
Collagen protein was determined by measuring hydroxyproline content in the samples. Added 10 mL 6 mol L−1 HCl and 3–4 drops of phenol to 4 g samples in the hydrolysate tube. Put the hydrolysis tube into the coolant and frozen for 3–5min. Input high purity nitrogen and vacuuming, and the samples was hydrolyzed to 22 h in a dry box at 110 °C. The hydrolyzed samples were fixed to 20 mL, and the content of hydroxyproline was determined by the automatic amino acid analyzer and external standard method.
The content of calcium and phosphorus in the deer bone, the DBP and the DBPCC were measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES optima 8000, Perkin Elmer Co., America). Appropriate amount of samples (accurate to 0.1 mg) was put into the polytetrafluoroethylene digestion tank, and then added 5 mL nitric acid. After the reaction was finished, the samples were sealed and put into the microwave digestion instrument. The digestion procedure is shown in Table 2. When the temperature was cooled to under 50 °C, put the tank into the wardrobe, and then wash the samples to the capacity bottle with ultrapure water. Dilute the volume to the scale with ultrapure water before analysis.
| Procedure | Temperature/°C | Holding time |
|---|---|---|
| 1 | 100 | 3 |
| 2 | 140 | 3 |
| 3 | 160 | 3 |
| 4 | 180 | 3 |
| 5 | 190 | 15 |
2.6 Determination of the amino acid components Amino acid components of deer bone powder, the DBP and the DBPCC were determined by the automatic amino acid analyzer (JASCO model, Hachioji-shi, Tokyo, Japan). The samples were hydrolyzed at 110 °C for 24 h using HCl (6 mol L−1) in vacuum-sealed ampoules and then being neutralized, evaporated and filtered with a glass filter before analyzed.
2.7 Spectroscopy analysis Ultraviolet–visible (UV) molecular absorption spectrometry is an effective method to evaluate some structural characterization through the dislocation and intensity change of UV absorption spectroscopy (Wang et al., 2017). Both the DBP and the DBPCC were dissolved in distilled water to a final concentration of 0.5 g L−1. Before sample determinations, the base line was set with distilled water and the samples were placed in quartz cuvettes with a path length of 10 mm for measurement. The ultraviolet (UV) spectra of the DBP and the DBPCC were recorded over the wavelength from 190 to 400 nm using a UV-vis spectrophotometer (SP-756P, Shanghai spectrum instrument Co., Ltd, China).
Fluorescence spectroscopy could be used to evaluate the interactions among protein and other molecules using a ShimadzuRF-5301 spectrometer. For determinations, 0.0307 g DBP powder was dissolved in distilled water with the addition of 0, 0.2, 0.6, 1.0, 1.4 and 1.8 mL of 0.1 mol L−1 CaCl2 respectively to form the DBPCC, and the total volume of all the sample solutions were controlled to 20 mL with distilled water. Then the fluorescence spectroscopy was recorded with fluorescence quartz cuvettes (1 cm) at room temperature. The excitation wavelength was 395 nm, and the emission wavelengths were recorded from 320 to 450 nm. The slit width of excitation and emission was 10 and 15 nm respectively, and the sampling interval was 1 nm.
The powders of the lyophilized DBP (2 mg) and DBPCC (2 mg) were fully mixed and grounded with 200 mg of dried KBr in an agate mortar and then be tableted, respectively. The mixture samples were loaded onto the Fourier transform infrared spectroscopy (FTIR) spectrograph and the spectrum was recorded using an infrared spectrophotometer (SHIMADZU IRAffinity–1) at the wavenumbers ranging from 4000 to 400 cm−1. A blank KBr pellet was used to subtracted the background and every sample was scanned 32 times at 4 cm−1 resolution.
2.8 Size distribution analysis Particle size, one of the important physical properties of composites, could be determined by the laser diffraction instrument through the measurement of particle volume distribution (Cai et al., 2014). The DBP and the DBPCC powder were dispersed in distilled water (RI = 1.360) and subjected to ultrasonic dispersing (FRQ-1008T, Front Ultrasonic Technology Co., Ltd., Hangzhou, China) for 15 min before the experiment. The size distribution of the DBP and the DBPCC were measured by a laser particle size analyzer (Zetasizer 3000HS, Malvern, UK) at room temperature. Particle size was computed in terms of the volume mean diameter (VMD) D[4,3], surface mean diameter (SMD) D[3,2], 10th percentile [d(0.1)], median [d(0.5)], 90th percentile [d(0.9)] and the distribution [span = (d(0.9) − d(0.1))/d(0.5)] (Cai et al., 2014, Michalski et al., 2005).
2.9 Determination of Molecular Weight Distribution The molecular weight distribution of the DBP and the DBPCC were determined by gel permeation chromatography (GPC) (Perkin Elmer Instrument Co., Ltd.) at room temperature. The mobile phase was acetonitrile: water (3:7,v/v). The injection volume was 100 µL and the running time was 45 min.
2.10 Morphology analysis for the DBP and the DBPCC The powders of the lyophilized DBP and DBPCC were mounted on the bronze stub, and sputter-coated with gold, respectively. Then the morphology of the samples were observed by a scanning electron microscope (XL-30 ESEM FEG Scanning Electron Microscope FEI COMPANY™) at an acceleration voltage of 10 kV.
The powders of the DBP and the DBPCC were dissolved in distilled water respectively and one drop of the solution were applied onto carbon-coated copper grids and dried at ambient temperature. Observation were analyzed by field emission transmission electron microscopy (Tecnai, G2, F20, S-TWIN) at 120 kV in a bright field.
2.11 Statistical analysis All experiments were conducted in triplicate and the results were expressed as means ± standard deviation (SD). Duncan's multiple range tests were used to analyze the variance (ANOVA) to determine the results' significance at p<0.05, using SPSS 19.0 software (SPSS Inc. Chicago, IL, USA).
3.1 Effects of the concentration of Ca2+ on the DBPCC As shown in Fig. 1(a), when the concentration of Ca2+ increased from 1.0 to 10.0 mmol L−1, the quantity of peptide chelated calcium increased from 18.08 mg g−1 peptide to 201.51 mg g−1 peptide notably (p<0.05). Meanwhile, with the increase of the Ca2+ concentration, the chelating rate increased first and stabilized until the Ca2+ concentration reached to 7.5 mmol L−1, and then the chelating rate decreased to 64.29 % when the Ca2+ concentration was 10.0 mmol L−1. Excessive calcium concentration (12.5 mmol L−1) was unfavorable to the chelation reaction since precipitation was formed in the reaction system. Therefore, the optimum Ca2+ concentration for the chelation reaction was 7.5 mmol L−1. The reason of Ca2+ induced the DBP precipitation was that calcium might shield the electrostatic repulsion of the charged protein molecules and form an antiparallel β-sheet structure stabilized by hydrogen-bonding, resulting in re-organization of protein secondary and tertiary structures (Zhang et al., 2012). A similar result also verified that increasing the CaCl2 concentration in Soy protein isolate solution could induce protein molecule aggregation and form the large precipitates (Zhang et al., 2012).
Effects of the DBP concentration (a), the concentration of Ca2+ (b), pH value (c), the temperature (d), and the reaction time (e) on the chelating rate and the quantity of peptide chelated calcium during the process of the DBP chelating with Ca2+. Values in the same figure with different letters are significantly different (P < 0.05) for chelating rate (lowercases) or quantity of peptide chelated calcium (uppercases), respectively.
3.2 Effects of the concentration of the DBP on the DBPCC With the increase of the DBP concentration, the chelating rate gradually rose and then stabilized. The quantity of peptide chelated calcium increased from 146.56 mg g−1 peptide to 163.62 mg g−1 peptide, and then decreased when the DBP concentration reached 2.30 mg mL−1 (Fig 1(b)). This phenomenon might be caused by the changes in the viscosity of the substrate. Increasing DBP concentration could increase the viscosity of the reaction system, so it was not conducive to get adequate contact between the DBP and Ca2+, and further affected the reaction results. When the concentration of the DBP was lower than 1.535 mg mL−1, the chelating rate became low, indicating that Ca2+ were excessive in the reaction system. With the concentration of the DBP reached more than 1.535 mg mL−1, the chelating rate was stable, and the DBP gradually became excessive, resulting in the decrease of the quantity of peptide chelated calcium. The low chelating rate would lead to the waste of Ca2+, and the low level of the quantity of peptide chelated calcium would lead to the reduction of calcium content in the DBPCC. Therefore, the optimal concentration of the DBP was 1.535 mg mL−1.
3.3 Effects of the temperature on the DBPCC As shown in Fig. 1(c), the chelating rate and the quantity of peptide chelated calcium kept stabilized from 30 °C to 40 °C, and then decreased significantly when the temperature was higher than 50 °C. Thus, the appropriate binding temperature was selected to be 40 °C. The chelating rate and the quantity of peptide chelated calcium at 40 °C was 68.32 % and 163.55 mg g−1 peptide, respectively. The change of temperature would affect the reaction rate and cause the stability constants of the complexes to change. These results proved that the DBPCC could be produced and maintained stable at 40 °C, so it would not become thermal denaturation in human body.
3.4 Effects of the reaction time on the DBPCC The chelation of the DBP and Ca2+ was such a fast reaction that the influence of chelating time was not significant after 15 min. After reacting for 15 min, the chelating rate and the quantity of peptide chelated calcium reached a stable value of 68.08 % and 160.05 mg g−1 peptide (Fig 1(d)), respectively. The chelating rate and the quantity of peptide chelated calcium did not decrease as the chelating time increased under the condition of pH 7.0 and 40 °C, which demonstrated that the DBP and Ca2+ had formed stable complexes. (Xixi et al., 2015) had shown that the chelation of peptide and calcium was rapid and could be balanced within half an hour at 30 °C, which was identical to the experimental results.
3.5 Effects of the pH value on the DBPCC As shown in Fig. 1(e), the chelating rate and the quantity of peptide chelated calcium changed significantly at various chelating pH value. The chelating rate and the quantity of peptide chelated calcium increased remarkably when the pH value increased from 3.0 to 7.0. The increase of pH value from 3.0 to 7.0 leaded to the decrease of hydrogen ions in the solution, so there were less hydrogen ions competed with calcium to bind electron-donating groups, which was beneficial to the formation of the DBPCC. At pH 7.0, the chelating rate and the quantity of peptide chelated calcium reached the maximum value of 69.70 % and 161.72 mg g−1 peptide, respectively. When the pH value was more than 7.0, the chelating rate and the quantity of peptide chelated calcium expressed a downtrend, and the precipitation obviously appeared in the solution at pH 11. In basic conditions, the excessive hydroxide ions would react with calcium to produce hydroxide precipitates, which would affect the chelation reaction (Lin et al., 2015). Therefore, the reacting condition was determined at pH 7.0.
3.6 The optimal preparation condition for the DBPCC As the chelation reaction progressed, calcium combination sites in the DBP would reacted with the added Ca2+ to form the DBPCC, which was mainly influenced by the concentration of the DBP, the concentration of Ca2+, pH value, the temperature, and the reaction time. According to the effect results of the factors, the chelation reaction conditions including 1.535 mg mL−1 DBP, 7.5 mmol L−1 CaCl2 at pH 7.0 and 40 °C for 15 min were performed to prepare the DBPCC to further characterize the properties. The work of (Choi et al., 2005) also showed that phosvitin purified from egg yolks could incubate with Ca2+ under ileum conditions (pH 7.0, 37 °C), and the phosvitin peptides were effective on enhancing the bioavailability of calcium.
3.7 Chemical analysis The chemical composition of deer bone powder, the DBP and the DBPCC were shown in Table 3. The deer bone powder was composed of 22.24±0.09 % protein, 9.18±0.07 % lipid, 7.53±0.05 % moisture and 54.23±0.12 % ash. As for the inorganic portion, deer bone powder has a high content of calcium (25.04±0.03 %) and phosphorus (12.08±0.02 %) with the mole ratio of Ca/P was 1.60, which was same as that of mammalian (1.68) and human (1.69) ratios (Jung et al., 2005). After the deer bone powder was hydrolyzed to the DBP, the content of calcium and phosphorus in the DBP decreased significantly, which indicated that the DBP had a high purity. Thus, the effect of calcium and phosphorus impurities in the DBP on the chelation reaction between the DBP and Ca2+ was small. The DBP contained 0.3859±0.0035 % calcium, and after the chelation reaction, the calcium content of the DBPCC increased to 9.45±0.03 %, indicating that calcium had combined to the DBP and formed the DBPCC. The phosphorus content of the DBPCC was 0.70±0.04 %, which was significantly higher than that of the DBP (0.2064±0.0020 %). That was probably because during the process of washing the chelating products by alcohol, some of the amino acids or peptides with no phosphorous were dissolved by alcohol and removed. The peptides containing phosphorus were precipitated in alcohol in the form of DBPCC, resulting in the high concentration of phosphorus. This phenomenon demonstrated that the phosphate group in the DBP probably was the main part of the DBP binding to Ca2+. In deer bone powder, the DBP and the DBPCC, collagen was present at 46.76 %, 35.18 % and 20.8 % of total protein respectively, indicating that both collagen and non-collagen protein could chelate with calcium ions.
| Chemical | Content | ||
|---|---|---|---|
| Deer bone powder / % | DBP/ % | DBPCC/ % | |
| Protein | 22.24±0.09 | 65.6±0.13 | 49.9±0.08 |
| Collagen | 10.38±0.12 | 23.08±0.10 | 10.38±0.12 |
| Collagen/Protein | 46.76 | 35.18 | 20.8 |
| Lipid | 9.18±0.07 | 3.1±0.06 | 5.4±0.09 |
| Moisture | 7.53±0.05 | 10.6±0.05 | 4.3±0.07 |
| Ash | 54.23±0.12 | 15.8±0.09 | 21.5±0.11 |
| Ca | 25.04±0.03 | 0.3859±0.0035 | 9.45±0.03 |
| P | 12.08±0.02 | 0.2064±0.0020 | 0.70±0.04 |
Data presented in the table were expressed as mean ± SD (n=3).
3.8 Amino acid components The function of protein or peptide depends mainly on its amino acid components. The amino acid components of deer bone powder, the DBP and the DBPCC are shown in Table 4. The DBP was rich in Gly (22.44 %), Glu (12.77 %), Pro (10.36 %), Ala (9.88 %), Arg (8.77 %) and Asp (6.84 %). After chelating with Ca2+, the DBPCC had high content of Pro (26.73 %), Leu (8.38 %), Asp (7.64 %) and Ala (6.42 %), which had been reported to be mainly components of calcium-chelating salmon ossein oligopeptides (SOOP-Ca) (Liu et al., 2015). The four kinds of amino acids could be regarded as the major sites of calcium combination and were probably related to the ability of peptide chelated calcium to some extent (Lin et al., 2015). As shown in Table 4, the content of negatively charged amino acids and positively charged amino acids in the DBP were 19.61 % and 14.88 %, respectively. After chelating with Ca2+, the content of negatively charged amino acids and positively charged amino acids in the DBPCC changed to 9.74 % and 13.69 %, respectively. Moreover, the DBPCC had a higher proportion of hydrophobic amino acids (57.18 %) than the DBP (34.37 %). These results indicated that the carboxyl group of negatively charged amino acids (Glu and Asp) (Lin et al., 2015), the positively charged residues (Lys, Arg and His) and hydrophobic amino acids residues (Liu et al., 2013) played an important role in calcium binding.
| Amino acid | Composition (mg/100mg of all amino acids) | ||
|---|---|---|---|
| Deer bone powder | DBP | DBPCC | |
| Asp | 8.97 | 6.84 | 7.64 |
| Thr | 4.59 | 3.00 | 4.32 |
| Ser | 4.31 | 3.80 | 4.12 |
| Glu | 1.71 | 12.77 | 2.10 |
| Pro | 29.85 | 10.36 | 26.73 |
| Gly | 4.94 | 22.44 | 5.38 |
| Ala | 6.24 | 9.88 | 6.42 |
| Cys | 0.18 | 0.17 | 0.21 |
| Val | 4.71 | 3.30 | 5.15 |
| Met | 0.50 | 1.13 | 0.64 |
| Ile | 3.56 | 1.75 | 3.68 |
| Leu | 8.01 | 4.82 | 8.38 |
| Tyr | 4.26 | 1.74 | 5.36 |
| Phe | 5.55 | 3.12 | 6.18 |
| Lys | 5.76 | 4.60 | 6.05 |
| NH3 | 0.00 | 0.00 | 0.00 |
| His | 1.98 | 1.51 | 2.21 |
| Trp | 0.00 | 0.00 | 0.00 |
| Arg | 4.90 | 8.77 | 5.42 |
| negatively charged amino acids (Glu and Asp) | 10.68 | 19.61 | 9.74 |
| positively charged amino acids (Lys, Arg and His) | 12.64 | 14.88 | 13.69 |
| hydrophobic amino acids | 58.42 | 34.37 | 57.18 |
| essential amino acids | 32.69 | 21.72 | 34.41 |
| aromatic amino acids (Phe, Tyr and Trp) | 9.81 | 4.86 | 11.54 |
Data presented in the table were expressed as mean ± SD (n=3).
It is reported that there was a positive correlation between carboxyl groups and calcium binding ability, while the imidazolyl groups was not important for calcium binding (Bao et al., 2008). One notable feature was that the amino acid components of deer bone powder and the DBPCC were in good agreement with each other. The essential amino acids in deer bone powder and the DBPCC accounted for 32.69 % and 34.41 % respectively, which confirmed that the DBPCC had a high nutritional value and it could be a good calcium fortified supplement.
3.9 Ultraviolet spectrum The UV absorption spectra of the DBP presented an obvious difference from that of the DBPCC (Fig. 2). The DBP had a strong absorption peak at 219 nm, which conformed to the characteristics of carbonyl group and amide bond, respectively (Lin and Liu, 2006). After chelating with Ca2+, the UV band and intensity of the DBPCC obviously shifted.
UV spectra of the DBP and the DBPCC over the wavelength range from 190 to 400 nm.
The maximum absorption peak of the DBPCC red shifted from 219 nm to 210 nm compared to the DBP (Fig. 2), which probably was due to the n→π* transitions of the carbonyl group in the peptide bond and the electron transition of calcium (Yu and Fan, 2012). The UV intensity (absorption) of the DBP was 3.736, and it decreased to 2.704 after the chelation reaction (Fig. 2), which indicated that carboxylate oxygen and imido nitrogen in the peptide bond combined with Ca2+ and changed the electron cloud of the amide bond (Zhao et al., 2015). The results of band shifts and intensity changes implied that the DBP could combine with Ca2+ through metal-binding sites and form the DBPCC.
3.10 Fluorescent spectrum As shown in Table 4, the aromatic amino acids content of the DBP and the DBPCC were 4.86 % and 11.54 %, respectively. Besides, the fluorescence spectra of the DBP containing different concentrations of CaCl2 are showed in Fig. 3 to evaluate the interactions among the DBP and Ca2+. After binding with Ca2+, fluorescence quenching would occur to peptides containing aromatic amino acids (Cai et al., 2016) and would contribute to the decrease of the fluorescence intensity. The decrease of fluorescence intensity is a classic indicator for peptide folding (Wu et al., 2012). In Fig. 3, the intensity of endogenous fluorescence at 363 nm obviously decreased from 579.369 to 420.275 as the Ca2+ concentration increased, which indicated that aromatic amino acids could chelate with Ca2+ and caused fluorescence quenching. Therefore, the results demonstrated that Ca2+ caused the DBP folding and conformation modification, and lead to the formation of the DBPCC. Another possible explanation was that the chromophores in the DBP reacted with Ca2+ and changed the energy in the excited state, which caused the decrease in fluorescence intensity (Wang et al., 2017).
Fluorescence spectra of the DBP with various concentrations of CaCl2 over the wavelength range from 320 to 450 nm.
3.11 Fourier transform spectrum (FT-IR) Fig. 4 shows the FT-IR spectra of the DBP and the DBPCC. Peptides mainly chelated with Ca2+ at sites including amide bonds, side chains and some carboxyl groups and terminal side amino groups (Liu et al., 2015). In the functional group region (4000–1300 cm−1), after the DBP chelating with Ca2+, the changes of the -NH stretching vibration were observed, and the red shift from 3327.21 cm−1 to 3288.63 cm−1 indicated that the -NH2 bond participated in the chelation reaction. The presence of organic material (C-H) was detected as peaks at 3072.60 cm−1, 2958.80 cm−1 and 2931.80 cm−1 in the FT-IR spectra of the DBP. It appeared as low intensity peaks at 2976.16 cm−1, 2908.65 cm−1 and 2841.15 cm−1 when the DBP combined with Ca2+, indicating that the chelation reaction had affected the C-H stretching vibration. In the fingerprint region (1300–600 cm−1), the spectra of the DBP exhibited a strong band at 1652.99 cm−1 (amide I band), corresponding to -C=O stretching vibrations. It appeared as two peaks (1652.99 and 1668.43 cm−1) when combined with Ca2+, indicating that the -COO− group participated in the covalent bonding to the metal ions (Lin et al., 2015). The peak (1404.18 cm−1) for the symmetric vibration of the -COO− carboxylate group shifted to a higher frequency (1417.68 cm−1) in the spectrum of the DBPCC, suggesting that ACOOA carboxylate group probably combined with Ca2+, turned into ACOOACa (Wang et al., 2017).
Fourier transform infrared (FTIR) spectra of the DBP and the DBPCC in the region from 4000 to 400 cm−1.
Furthermore, the peaks of 1244.09 cm−1 and 1201.65 cm−1 in the spectra of the DBP, which belonged to amide III, changed from two peaks to a single-peak at 1246.02 cm−1 when combined with Ca2+, causing by the N–H bending vibration and C–N stretching vibration. Additionally, the peak observed at 1161.15 cm−1 decreased and moved towards 1199.72 cm−1 when the DBP combined with Ca2+. A reasonable explanation was that the DBP bound with Ca2+ and formed C–O–Ca (Cai et al., 2016). In the range of 1100 cm−1 ∼ 1000 cm−1, two absorption peaks shifted from 1082.07 cm−1 to 1080.14 cm−1 and from 1033.85 cm−1 to 1037.70 cm−1 when combined with calcium, which arose from the changes of C–O stretching and −OH deformation vibrations (Zhang-yan et al., 2011). Moreover, the peak observed at 920.05 cm−1 disappeared when combined with Ca2+, which probably arose from the changes of out of plane deformation vibration of O-H and the changes of phosphate groups in the DBP. The results of the FT-IR indicated that calcium bound to the DBP primarily through carboxyl oxygen atoms and amino nitrogen atoms interaction sites. Changes of the FT-IR spectrum (Fig. 4) proved that the DBPCC was a typical peptide-chelated calcium.
3.12 Particle size distribution The particle size distributions of the DBP and the DBPCC are shown in Fig. 5. The standard mean diameters [D(4,3) and D(3,2)] and distribution information [d(0.1), d(0.5), and d(0.9)] are all shown in Table 5. The volume mean diameter (VMD) of the DBP was determined to be 99.25±13.83 nm, and the VMD of the DBPCC was 181.25±2.68 nm. The d(0.5) value of the DBP (77.84±4.68 nm) was estimated to be smaller than that of the DBPCC (140.52±5.29 nm). These results indicated that there were not only intramolecular interactions, but also intermolecular interactions existing in the chelation reaction between the DBP and Ca2+. Moreover, the DBPCC were proved to be a compact nano-composite through the particle size distribution.
Particle size distributions of the DBP and the DBPCC
| Samples | d(0.1) | d(0.5) | d(0.9) | D[4,3] | D[3,2] | Span |
|---|---|---|---|---|---|---|
| DBCP | 9.23±0.71 | 77.84±4.68 | 208.11±29.82 | 99.25±13.83 | 14.27±0.76 | 2.56±0.21 |
| DBCPCC | 21.73±1.07 | 140.52±5.29 | 403.32±1.07 | 181.25±2.68 | 30.37±1.90 | 2.72±0.11 |
Values are expressed as mean ± standard deviations, n=3.d(0.1), d(0.5), and d(0.9) are the granule sizes at which 10, 50, and 90 % of all the granules by volume are smaller, respectively. D(3,2) is the surface area-weighted mean diameter. D(4,3) is the volume-weighted mean diameter.
3.13 Determination of Molecular Weight Distribution Molecular weight distribution is an important factor that reflecting the calcium-binding capacity of peptides (Lin et al., 2015). The molecular weight distribution of the DBP and the DBPCC were shown in Fig. 6 and Fig. 7. The DBP was mainly distributed under 1000 Da, which accounted for 86 %, whereas the molecular weight fraction of more than 1000 Da was only 14 % (Fig. 6). This result suggested that the process of enzymatic hydrolysis reduced the molecular weight of the deer bone protein, and the low molecular weight peptides might play an important role in the chelation reaction. It has been shown that soluble bone collagen peptide with molecular weight under 5 kDa had higher calcium binding capacity (Yong et al., 2011). Furthermore, other low molecular weight peptides had also been identified to possess high calcium affinity, such as wheat germ peptides ranging from 1000 to 180 Da (Liu et al., 2013) and Schizochytrium sp. protein hydrolysate less than 2000 Da (Lin et al., 2015). The molecular weight distribution of the DBPCC was ranging from 2000 Da to 6000 Da, and most of them were between 2000 to 3000 Da. The molecular weight of the DBPCC was estimated to be larger than that of the DBP, which was correspond to the particle size distribution (Fig. 5). These results indicated that Ca2+ was probably combined with functional binding sites from two or more peptides, and there were both intramolecular interactions and intermolecular interactions between the DBP and Ca2+. (Zhao et al., 2014) has also demonstrated that calcium ion was surrounded by the carboxyl and amino groups of Gly-Tyr to form the Gly-Tyr-Ca chelate.
Molecular weight distribution of the DBP.
Molecular weight distribution of the DBPCC.
3.14 Morphological characteristics The microstructures of the DBP and the DBPCC are illustrated in Fig. 8. SEM micrographs of the DBP and the DBPCC in multiples of 2000 are displayed in Fig. 8(a) and Fig. 8(b), Fig. 8(c) and Fig. 8(d) displayed the TEM image of the DBP and the DBPCC. From Fig. 8(a), the DBP presented a loose structure with many irregular size holes. After chelating with Ca2+, the DBPCC showed a dense structure with a lot of white rod-like shape particles (Fig. 8(b)). As shown in Fig. 8(c) and Fig. 8(d), the images of the DBP and the DBPCC could visually reveal the particle morphology. From Fig. 8(c), the TEM image of the DBP exhibited a diffuse shape and these granule-like structure placed irregularly in the visualized area. The intermolecular hydrogen bond in the aqueous solution caused the peptide chain aggregation and formed these granules (Lin et al., 2015). After chelating with Ca2+, the DBPCC presented shuttle-shaped structures with a regular and dense crystalline character (Fig. 8(d)).
SEM imagines (5000×) of the DBP (a) and the DBPCC (b) as well as the TEM imagines of the DBP (c) and the DBPCC (d). The part of the break in (a) might be caused by drying.
The remarkable structural changes might be caused by two reasons. First of all, carboxyl groups and amino groups in the DBP chelated with calcium and formed “bridging role”, so the DBP properties were changed (Wang et al., 2017). Secondly, the added Ca2+ shielded the negative charges on polypeptide chains and formed a “salt bridge” to induce protein aggregation (Chen et al., 2014). The similar phenomenon was also observed by (Peng et al., 2017). All of the structural changes between the DBP and the DBPCC proved that the DBP chelated with Ca2+ to form the DBPCC, so the DBP and the DBPCC were two completely different substances.
In this work, a novel calcium supplement DBPCC was prepared through the chelation reaction between the DBP and Ca2+. The DBP exhibited good calcium-binding capacity when reacted with Ca2+ at the optimal chelation conditions. The carboxyl oxygen atoms and amino nitrogen atoms participated in the combination between the DBP and Ca2+ to form the DBPCC. The structure of the DBP and the DBPCC were totally different since peptides folding and conformation modification occurred to the DBP after adding Ca2+, causing the size distribution of the DBPCC to become larger than the DBP. The molecular weight distribution of the DBPCC was mainly ranging from 2000 Da to 3000 Da, larger than that of the DBP (<1000 Da). The DBPCC also showed relatively high stability under ileum condition, indicating that it could be a well absorbed calcium supplement with high nutritional value. This study not only provides a novel calcium supplement but also helps to find an effective method to provide high value-added deer bone products.
Acknowledges This work was financially supported by National Natural Science Foundation of China (Project number 31571857 and 31371804). The authors also wish to thank the Changchun Institute of Applied Chemistry Chinese Academy of Sciences and Lishi Deer Farm in Changchun, Jilin Province.
