Preparation and Evaluation of the Chelating Nanocomposite

Article pubs.acs.org/JAFC

Preparation and Evaluation of the Chelating Nanocomposite Fabricated with Marine Algae Schizochytrium sp. Protein Hydrolysate and Calcium Jiaping Lin,†,§ Xixi Cai,†,‡,§ Mengru Tang,† and Shaoyun Wang*,† †

College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian 350108, People’s Republic of China College of Chemistry, Fuzhou University, Fuzhou, Fujian 350108, People’s Republic of China

‡

ABSTRACT: Marine algae have been becoming a popular research topic because of their biological implication. The algae peptide-based metal-chelating complex was investigated in this study. Schizochytrium sp. protein hydrolysate (SPH) possessing high Ca-binding capacity was prepared through stepwise enzymatic hydrolysis to a degree of hydrolysis of 22.46%. The nanocomposites of SPH chelated with calcium ions were fabricated in aqueous solution at pH 6 and 30 °C for 20 min, with the ratio of SPH to calcium 3:1 (w/w). The size distribution showed that the nanocomposite had compact structure with a radius of 68.16 ± 0.50 nm. SPH was rich in acidic amino acids, accounting for 33.55%, which are liable to bind with calcium ions. The molecular mass distribution demonstrated that the molecular mass of SPH was principally concentrated at 180−2000 Da. UV scanning spectroscopy and Fourier transform infrared spectroscopy suggested that the primary sites of calcium-binding corresponded to the carboxyl groups, carbonyl groups, and amino groups of SPH. The results of fluorescent spectroscopy, size distribution, atomic force microscope, and 1H nuclear magnetic resonance spectroscopy suggested that calcium ions chelated with SPH would cause intramolecular and intermolecular folding and aggregating. The SPH-calcium chelate exerted remarkable stability and absorbability under either acidic or basic conditions, which was in favor of calcium absorption in the gastrointestinal tracts of humans. The investigation suggests that SPH-calcium chelate has the potential prospect to be utilized as a nutraceutical supplement to improve bone health in the human body. KEYWORDS: Schizochytrium sp. protein hydrolysate-calcium chelate, nanocomposite, fabrication, characterization

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INTRODUCTION Marine algae, which have traditionally formed part of the Oriental diet, especially in China, Japan, and Korea, have been becoming a hot research topic because of their biological substances.1 In Western countries, their major use has traditionally concentrated on the extraction of compounds used by pharmaceutical and food industries as a source of phycocolloids and thickening and gelling agents (production of agar, alginate, carrageenan, etc.).2,3 Nowadays, the field of marine natural food products has become more sophisticated. Schizochytrium sp., also known as Schizochytrium aggregatum, belongs to marine fungi of Eumycota, Oomycetes, Saprolegniales, and Thraustochytriaceae. Schizochytrium sp. has been used as a fundamental source of human foods and drugs in traditional Chinese medicine for centuries. Schizochytrium sp. cells possess a large number of active substances beneficial to humans, such as pigment, fat, unsaturated fatty acids, proteins, polysaccharides, squalene, etc.4 A great number of researchers principally focused on the industrial production of docosahexaenoic acid for Schizochytrium sp. studies.5,6 Nevertheless, in addition to the high content of fat, Schizochytrium sp. also contains a big amount of protein, which is determined to be 41.29 ± 0.51% (dry weight). However, the defatted Schizochytrium sp. is mostly used for biological baits, and little research on the Schizochytrium sp. protein has been reported. Calcium is an essential nutrient required by the human body and participates in all processes of life, which plays a particularly significant role in maintaining the structure and physiological © 2015 American Chemical Society

function. A series of reports proved that calcium deficiency was linked with the development of disease, including osteopenia, osteoporosis, arterial hypertension, and kidney stones etc.7,8 In recent years, ionized calcium has been the primary calcium supplement for humans9 since the calcium in the body could be controlled to maintain the advisible content through the intake of calcium. The casein-ophosphopeptides (CPPs), phosphorylated peptides derived from dairy, have shown considerable effect in promoting calcium absorption and are now extensively used for mineral binding, whereas the whey peptides, obtained from proteolytic digestion, have proved to have effective capacity in incorporating with calcium iron.10,11 CPPs, as the animal-based modified peptides, chelated with calcium mostly through the negatively charged phosphate groups, whereas the SPH, which belongs to natural peptide from proteolytic digestion and not phosphorylated peptide, chelated with calcium mostly through the side-chain carboxylates. It is reported that the peptide-calcium chelate is easier to absorb and utilize on account of its unique chelating system and transport mechanism, and it could be absorbed by the mucosal cells in the gastrointestinal tract but would not be destroyed in the acid and alkali gastrointestinal environment.12 As a result, it would not be affected by phytic acid, oxalic acid, and phosphoric Received: Revised: Accepted: Published: 9704

August 14, 2015 October 16, 2015 October 24, 2015 October 25, 2015 DOI: 10.1021/acs.jafc.5b04001 J. Agric. Food Chem. 2015, 63, 9704−9714

Article

Journal of Agricultural and Food Chemistry

20 °C, for 10 min to remove the insoluble calcium phosphate salts. The calcium content in the supernatant was determined using the colorimetric method, and the absorbance at 570 nm was measured after introducing the working solution to the sample. Determination of the Degree of Hydrolysis. The degree of hydrolysis (DH) was monitored using the formaldehyde titration method.15 Five milliliters of hydrolysate sample was placed in a 150 mL beaker. After adding 60 mL of distilled water, the pH was adjusted to 8.2 with a pH meter using 0.01 mol/L of standard NaOH solution under constant agitation. Twenty milliliters of formaldehyde solution (pH 8.2) was added into the above solution, and the titration volume of 0.01 mol/L of standard NaOH solution was recorded when the pH was titrated to 9.2. The DH in the sample was calculated as follows:

acid of other foods. Once entering into the blood, the peptidecalcium chelate could continuously dissociate in the body, thereby extending the calcium releasing cycle and increasing the calcium bioavailability. Cai et al.13 reported that the calcium absorption of the chelate of whey protein hydrolysate and calcium in Caco-2 cells was markedly higher than that of calcium gluconate and CaCl2 in vitro, manifesting the potential increase in calcium bioavailability. The aim of this study was to prepare the nanocomposites of Schizochytrium sp. protein hydrolysate (SPH) chelated with calcium ions to investigate the characterization of nanocomposites and calcium releasing percentage to explore the possible cheating mechanism. The study could provide a new train of thought of the Schizochytrium sp. protein for the potential application as the algae-based dietary supplements of peptide-calcium nanocomposite.

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DH =

MATERIALS AND METHODS

n=

Materials. Schizochytrium sp. was kindly provided by Fisheries Research Institute of Fujian, China. The commercial protease, Alcalase (EC. 3.4.21.62, 2.2 × 105 U/g) and Flavourzyme (EC. 3.4.11.1, 7.8 × 104 U/g) were purchased from Novo (Novozymes, Denmark). All chemical reagents were of analytical grade and commercially available. Extraction of Schizochytrium sp. Protein. The method of alkali extraction and acid precipitation was used in this study to prepare Schizochytrium sp. protein (SP). The Schizochytrium sp. was ground to a powder (sieved through a 50 mesh sieve). One percent (w/v) Schizochytrium sp. powder in 0.39 mol/L NaOH solution was stirred at 90 °C for 30 min and then centrifuged at 10,000 rpm, 4 °C, for 20 min. The supernatant was adjusted to pH 3.0 by 6 mol/L HCl solution and kept for 30 min. The mixture was centrifuged at 10,000 rpm, 4 °C, for 15 min. The SP in the precipitation was lyophilized for further enzymatic hydrolysis. Optimization of Hydrolysis Conditions. In order to prepare the calcium-binding peptides, the hydrolysis conditions of SP were first optimized. The calcium-binding peptides were prepared through stepwise enzymatic hydrolysis by Alcalase and Flavourzyme, respectively, with the calcium-binding activity serving as the principal indicator and the degree of hydrolysis as the auxiliary index to optimize the hydrolysis conditions. The freeze-dried SP was first dissolved in distilled water at different substrate concentrations of 1, 3, 5, 7, and 9% (w/w), and the ratio of Alcalase to SP was 2, 4, 6, 8, and 10% (w/w), respectively. To investigate the time effects on both calcium-binding activity and degree of hydrolysis, the reaction solution was carried out in a water bath shaking at pH 9.0 and 50 °C for different time (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, 10.0, and 12.0 h, respectively). The sample solution was heated at 100 °C for 10 min to inactivate the protease, and then the pH was adjusted to pH 6.0. Flavourzyme (enzyme/substrate ratio was 4, 6, 8, 10, and 12%, w/w) was subsequently added to get further hydrolysis at 45 °C for 0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, and 8.0 h, respectively. The sample solution was boiled for 10 min to inactive Flavourzyme and then cooled to room temperature. The reaction solution was centrifuged at 10,000 rpm for 20 min at 4 °C, and then the supernatant named SPH was collected and lyophilized for further analysis and the following preparation of the SPH-calcium chelate. Analysis of the Calcium-Binding Activity. The calcium-binding activity was measured with ortho-cresolphthalein complexone reagent according to the method described by Wang et al.14 with some modifications. The calcium-binding activity was expressed as calcium binding amount per milligram peptides (w/w). After 1 mL of 9 mmol/L CaCl2 and 2 mL of 0.2 mol/L sodium phosphate buffer (pH 8.0) were adequately mixed, 1 mL of 1 mg/mL SPH was added to make a competitive environment since SPH-Ca was more stable than calcium phosphate salts. A SPH-free control experiment was conducted, and deionized water in the same volume instead of SPH was used in a blank experiment. The reaction solution was stirred at 37 °C for 2 h in a shaker (140 rpm), and the pH was maintained at 8.0. Then, the mixture was centrifuged at 10,000 rpm,

(n − n0) × 100 htot

(1)

V ΔV × C × tot W V

(2)

1 × Pro % 110

(3)

htot =

where n is millimoles of free amino per gram of protein after hydrolysis (mmol/g), and n0 is millimoles of free amino per gram of protein before hydrolysis (mmol/g). htot is total peptide bonds in the protein of raw material. ΔV is the D-value of the wasted volume of standard NaOH solution between the sample and protein of raw material, Vtot is the total volume of the hydrolysate, and V is the volume of the hydrolysate to titrate. C is the concentration of the standard NaOH solution (mol/L), and W is the gram number of the raw material. Pro % is the protein concentration of sample, and 110 is the average molecular mass of amino acids. Analysis of Amino Acid Composition. The lyophilized samples of SPH were hydrolyzed at 110 °C for 24 h using 6 mol/L of HCl, and the amino acid composition of SPH was analyzed with a High Speed Amino Acid Analyzer Model L-8900 (Hitachi, Japan) at the Instrumental Analysis Center of Jiangnan University, China. Determination of Molecular Mass Distribution. The molecular mass distribution of SPH was determined utilizing high performance liquid chromatography (HPLC), and 10 mL of sample at 0.5 mL/min was assembled with a Waters 650E Advanced Protein Purification System (Waters Corporation, Milford, MA, USA). Fabrication of SPH-Calcium Chelate. The freeze-dried SPH was dissolved in distilled water to a concentration of 1 mg/mL, and CaCl2 solution was introduced subsequently at a ratio of SPH to calcium 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1 (w/w) at various pH (3, 4, 5, 6, 7, and 8), respectively. The reaction solution was placed in a shaking water bath with constant agitation (140 rpm) at 30 °C for 20 min. Then, absolute ethanol was introduced continuously until the final solution volume was 10 times larger than that of the incipient solution, and all of the chelates were deposited and then centrifuged at 10,000 rpm for 20 min at 4 °C. The precipitates, named as SPH-calcium chelates, were collected and freeze-dried for further investigation. Ultraviolet Spectroscopy. Based on the displacement and intensity change of the ultraviolet (UV) spectra, the functional mechanism between SPH and calcium ion could be speculated. SPH solution (50 μg/mL) was first prepared and adjusted to pH 7.0. Then the SPH-calcium chelate was subsequently obtained by constantly adding 0, 0.5, 0.5, 1.0, 1.0, 1.0, and 1.0 μL of 2 mol/L CaCl2 to the sample solution, every 10 min. The UV spectra of SPH and its calcium chelate were recorded over the wavelength range from 190 to 400 nm using a UV−vis spectrophotometer (UV-2600, UNICO Instrument Co. Ltd., Shanghai, China) with the method described by Chen et al.16 Fluorescence Spectroscopy. Fluorescence spectra analysis of SPH-calcium chelate could further monitor whether Ca ions result in the conformational changes of the SPH by calcium chelation using a Hitachi F-4600 fluorescence spectrophotometer (Hitachi Co., Japan). An excitation wavelength of 280 nm and emission wavelengths between 295 and 500 nm were recorded. The slit width of excitation and emission 9705

DOI: 10.1021/acs.jafc.5b04001 J. Agric. Food Chem. 2015, 63, 9704−9714

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Journal of Agricultural and Food Chemistry were 10 nm, and PMT volts of the machine was 650 V. The preparation method of sample was the same as that of ultraviolet spectroscopy. Fourier Transform Infrared Spectroscopy (FTIR). A lyophilized powdered sample (1 mg) was fully mixed and ground with 100 mg of dried KBr (Spectral) in an agate mortar. After tableting, the mixture sample was loaded onto the Fourier transform infrared spectroscopy (FTIR) spectrograph. All FTIR spectra were recorded at room temperature by an infrared spectrophotometer (360 Intelligent, Thermo Nicolet Co., USA) from 4000 to 400 cm−1. For each spectrum, 64 scans were acquired at 4 cm−1 resolution. The peak signals in the spectra were analyzed using OMNIC 8.2 software (Thermo Nicolet Co., Madison, WI, USA). Size Distribution. The sizes distribution of the SPH and SPHcalcium chelate was determined by a laser particle size analyzer (Zetasizer 3000HS, Malvern, UK). One milligram of dried sample was dissolved in 1 mL of deionized water (RI = 1.330) and passed through a 0.22 μm filter membrane before the experiment. For test conditions, the cuvette size was 1 cm of polystyrene pool, and the pitch in a pair of 0.45 cm2 platinum electrodes was 0.4 cm. The measurement temperature was 25 °C, and the time of temperature equilibrium was 2 min. Atomic Force Microscopy. The morphological changes between SPH and SPH-calcium chelate were analyzed using atomic force microscopy (AFM, TM-AFM 5500, Agilent Technologies, USA). Two microliters of SPH/SPH-calcium chelate (50 μg/mL) was placed on the surface of freshly cleaved mica, diffused, and dried with a stream of nitrogen at room temperature. The images were subsequently obtained adopting tapping mode, employing microfabricated silicon cantilevers tips with a 322 kHz of resonance frequency and a 40 N/m of spring constant. 1 H Nuclear Magnetic Resonance Spectroscopy (NMR). The structural changes of the carbon bond and hydrogen bond of SPH and SPH-calcium chelate were analyzed and compared using a Bruker Avance III spectrometer (Bruker Biospin, Rheinstetten, Germany). The SPH and SPH-calcium chelate (0.5 mg) were dissolved in 500 μL of deionized water, and then 50 μL deuterium oxide (D2O) was added after the pH value of the solution was regulated to pH 6.5. The samples were carefully transferred into 5 mm NMR tubes for the test. Assay of Calcium Ion Releasing Percentage. SPH-calcium chelate and CaCl2 were dissolved in deionized water to a concentration of 50 μg/mL, and the releasing percentage of calcium ions was assayed at pH ranges of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0. After incubation in a water bath shaking with constant agitation (140 rpm) at 37 °C for 2 h, the solutions were centrifuged at 10,000 rpm for 10 min at 4 °C. The calcium content of the supernatant and the total calcium in the solution were measured using a colorimetric method with orthocresolphthalein complexone reagent. The calcium releasing percentage was calculated as follows: calcium releasing (%) =

calcium in supernatant × 100 total calcium in solution

Figure 1. Effects of enzymatic hydrolysis time (A), the ratio of Alcalase to SP (B), and substrate concentration (C) on the calcium-binding capacity and DH. All data are presented as the means ± SD in 3 independent experiments.

(4)

Statistical Analyses. All of the experiments were presented as the means (standard deviations, SDs) in triplicate. Statistical analysis was performed adopting SPSS 17.0 (SPSS, Chicago, IL, USA) and was done using Student’s t test. A confidence level of P < 0.05 was considered statistically significant.

ranged from 2 to 12% (w/w), the calcium-binding activity and DH of SP gradually went up and reached a constant at 10% (w/w) subsequently, and the maximum values of the calciumbinding activity and DH were 2.68 μg/mg peptide and 7.88%, respectively. With increasing concentration of substrate, the calcium-binding activity presented a significant tendency to decline, whereas the DH showed a downward trend after the first increase (Figure 1C). This phenomenon may be related to the viscosity of the substrate. Increasing substrate concentration could result in the increase of viscosity, so it is not conducive to get adequate contact between enzyme and substrate and further affect the reaction results.17 The experiment was conducted using the calcium-binding activity and the DH as the synchronous indicator. The hydrolysis conditions including 10% of E/S, 1% of substrate concentration at pH 9.0, and 50 °C for 8 h were taken to go ahead with the second-step of hydrolysis. The results of the second-step of hydrolysis under optimal conditions are shown in

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RESULTS AND DISCUSSION Optimization of Hydrolysis Conditions. In this study, the extraction rate of Schzochytrium sp. protein was 76.58 ± 0.04% through the method of alkali extraction and acid precipitation. As was expected, the calcium-binding activity and DH of SP increased with the persistent period of Alcalase hydrolysis (Figure 1A), which resulted in a typical curve. For the first 6 h, the calcium-binding activity gradually rose accompanied by a pronounced increase of the DH. Subsequently, the calciumbinding activity reached the maximum value of 2.91 μg/mg peptide at the 8th hour of hydrolysis, and the DH reached a stable value of 7.43% after 8 h. In Figure 1B, the ratio of Alcalase to SP 9706


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SPH was rich in glutamic acid (17.66%), aspartic acid (15.89%), leucine (9.96%), arginine (7.81%), and lysine (5.79%), which were well regarded as major sites of peptide-calcium chelating. The serine and threonine were central sites of metal ion combination and phosphorylation,18 and the content of serine and threonine in SPH was 5.79% and 3.93%, respectively. SPH also contained relatively more alanine residues (5.28%) that might be relative to the calcium chelate capacity to some extent. In Table 2, the acidic and basic amino acids accounted for Table 2. Amino Acid Compositions of SPH amino acids

content (%)

negatively charged amino acidsa positively charged amino acidsb hydrophobic amino acidsc essential amino acidsd aromatic amino acidse

33.55 15.17 34.71 37.71 7.99

a

Containing Glu and Asp. bContaining Lys, Arg, and His. cContaining Ala, Phe, Ile, Leu, Pro, Val, Try, and Met. dContaining Lys, Try, Phe, Met, Thr, Ile, Leu, and Val. eContaining Phe, Tyr, and Trp.

33.55% and 15.17%, respectively. Besides, the hydrophobic amino acids took up 34.71%, which were favorable to bind calcium.19 A number of previous reports have demonstrated that the carboxyl group of acidic amino acids (Glu and Asp), the imidazole group of His, and the negative/positive charges of the peptide play a greatly crucial role in calcium binding; they are therefore considered as main calcium binding sites.9,10,20 It is reported that the binding of calcium to the caseinophosphopeptides (CPPs) formed through proteolytic digestion occurs mostly through the negatively charged phosphate groups, whereas for the whey protein hydrolysate, binding principally depends on the side-chain carboxylates, which are not phosphorylated.20 Moreover, a high affinity calcium-binding peptide was reported to have a linear increase of calcium binding capacity accompanied by the augment of carboxyl content in different Asp and Glu contents of the peptides.10,19 Furthermore, it is reported that the positively charged residues (Lys and Arg) also play an indirect role of importance for calcium binding.19 Chen et al. reported that the amino acid sequence of calcium-binding peptide prepared from tilapia scale protein hydrolysate was Asp-Gly-Asp-Asp-Gly-Glu-Ala-Gly-Lys-Ile-Gly, which contained higher Asp and Glu.21 The Theragra chalcogramma backbone peptide could solubilize an analogous calcium content with CPPs in an in vitro calcium-binding assay.22 Therefore, the results of amino acid composition of SPH indicated that the carboxyl group of acidic amino acids and hydrophobic amino acids residues might contribute to the calcium chelate activity of SPH. Additionally, from the results we could consider that Schizochytrium sp. protein hydrolysate had a higher nutritional value, due to its higher content of the essential amino acid (37.71%, Table 2). Analysis of Molecular Mass Distribution. Molecular mass is a significant factor reflecting the hydrolysis of protein, which further reflects the bioactivity of protein hydrolysates.23 The result of the molecular mass distribution of SPH fractions was determined by HPLC as shown in Figure 3A. The molecular mass fractions of SPH were mainly distributed between 180 and 2000 Da, which accounted for 43.17% of the protein hydrolysates, whereas the molecular mass fraction of 6, the calcium-binding capacity decreased, which indicated that the hydroxyl began to form the hydroxide precipitate in the reaction solution and was not conducive to obtaining the peptide-calcium chelate.27 The chelating pH is an important parameter that affects the formation of chelate between peptides and microelements. In the acidic conditions, H+ would compete with metal ions to contend for electron-donating groups, which is favorable to form the peptidemicroelement chelate. In basic conditions, the excessive hydroxide ions would contend with electron-donating groups to compete for the metal ion to form a hydroxide precipitate.28 Ultraviolet (UV) Spectroscopy. Ultraviolet−visible molecular adsorption spectrometry (UV−vis) is principally produced by the transitions of the molecular valence electron between electronic energy levels, which is an investigative method that analyzes the substantial electronic spectra. The UV spectra of SPH showed an obvious difference from that of the SPH-calcium chelate (Figure 5). The SPH had a strong absorption peak at about 200 nm, which could be viewed as the characteristics of the carbonyl group, carboxyl group, and amide bond in the peptide. With increasing concentration of calcium chloride of 0, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mmol/L in the chelating procedure, the UV intensity of samples went up in a proper sequence, and the absorption peak of the samples shifted from 1.892 to 2.112 (Figure 5) due to the transition of the carbonyl group in the peptide bond from n → π* and the electron transition of calcium.29 Additionally, the oxygen atom of the carbonyl group and nitrogen of the amino group in the peptide bond could result

distribution of peptides. The molecular mass of wheat germ peptide possessing the high calcium affinity had been identified to be 180 to 1000 Da, which indicated the molecular mass of peptides has a significant role in calcium-binding capability.10 Jin et al. reported that the molecular mass of soluble bone collagen peptide which had a relatively high calcium combining capability was less than 5000 Da.24 A 1561 Da peptide isolated from hoki (Johnius belengerii) frame protein hydrolysates was confirmed to possess strong calcium-binding capacity. 25 Furthermore, other marine protein hydrolysates with molecular mass ranging from 1,000 to 3500 Da had higher calcium-binding capability as well.22,25,26 To sum up, higher calcium chelating capacity was likely to associate with the lower molecular mass distribution less than 2000 Da of SPH (Figure 3B). Fabrication of SPH-Calcium Chelate. The SPH-Calcium chelate was fabricated to further characterize the properties. In order to improve the efficiency of the fabrication of SPH-Ca chelate, the effects of chelating reaction pH and mass ratio of SPH to calcium were optimized. The mass ratio of SPH to calcium played a crucial role in the chelation reaction of SPH and calcium ions as shown in Figure 4A. When the ratio increased from 2:1 to 3:1 (w/w), the calcium-binding capacity highly notably increased, and the maximum value was 162.68 μg/mg of peptide. Subsequently, as the ratio increased, the calciumbinding capacity gradually slowed down. It is likely that excess peptides and lower calcium with the increasing proportion of peptides result in the gradually declining trend of calcium-binding capacity in the chelating reaction process after 3:1 (w/w). The mass of SPH-Ca chelate remarkably changed at various chelating pH, which was shown in Figure 4B. Within a certain pH range of 3−6, the calcium-binding capacity increased as the pH value rose. As the pH increased, the -NH2 and -COOH 9708


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for the transformation phenomenon could be that the chirality of the chromospheres (CO, −COOH) and auxochromes (−OH and -NH2) changed after the peptide bonded with a metal ion.31,32 All in all, the result of band shifts indicated that SPH bound with calcium ion and, as a result, formed a compact SPH-calcium chelate. Fluorescence Spectroscopy. Aromatic amino acids in a protein molecule, phenylalanine, tyrosine, and tryptophan, could emit fluorescence at a certain excitation wavelength. As shown in Table 2, the SPH contained a certain amount of aromatic amino acids (7.99%), which could generate endogenous fluorescence at an appropriate excitation wavelength. Therefore, the SPH and its calcium chelate were scanned using fluorescence spectroscopy to explore and identify the interactions among SPH and Ca ions in composite. As revealed in Figure 6, the fluorescence absorption band was dramatically decreased from 420.934 to 388.119 nm, which might be due to the formation of the chelate between SPH and metal ions to generate fluorescence quenching. Especially, the endogenous fluorescence obviously decreased when 1 mmol/L of CaCl2 was introduced to the SPH solution (Figure 6). Decreased fluorescence intensity was a classic indicator for peptide folding, indicating the apparent folding and structural modification of SPH.11,33 Previous study has shown that the Zn (II) combined with zinc-finger peptide could generate the folding of the peptide in the zinc-binding reaction.34 Moreover, the introduction of metal ions would induce the inherent fluorescence quenching of protein or peptide, particularly for the oligo-peptide without a thermodynamically stable structure in the reaction solution.35 With the calcium ion content increased, the declining extent of endogenous fluorescence was gradually weak. A potential interpretation was that the action sites of the peptide chain combined with the Ca ion led to the generation of the coordination bond and had no extra action sites of group subsequently. As a result, SPH was no longer involved in the chelate reaction, and the structure no longer changed, which showed an extremely weak effect in the fluorescence spectra of endogenous quenching. The calcium ions bound with SPH might cause fluorescence quenching, which contributed to the decrease in fluorescence intensity.36

Figure 4. Effects of mass ratio of SPH to calcium (A) and chelating reaction pH (B) on the calcium-binding capacity of SPH-Ca chelate. All data are presented as the means ± SD of 3 independent experiments.

in the changes of intensity and shift of characteristic absorption peak after combining with the calcium ion.30 The result of band displacements/shifts indicated that SPH could bind with calcium ion and form a SPH-calcium chelate. A reasonable explanation

Figure 5. UV spectra of SPH with different CaCl2 concentrations over the wavelength range from 190 to 400 nm. 9709


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Figure 6. Fluorescence spectra of SPH with different CaCl2 concentrations over the wavelength range from 295 to 500 nm.

Fourier Transform Infrared Spectroscopy. The spectrum changes of Fourier transform infrared spectroscopy could be observed when calcium ions chelate with the amino acid residues in peptides.37,38 Infrared spectra of SPH and SPH-calcium chelate were shown in Figure 7. After the addition of calcium,

Figure 8. Particle size distributions of SPH (A) and SPH-calcium chelate (B). All measurements were expressed as the means ± SD of 3 independent experiments.

in SPH belonged to amide III and shifted to 1318.82 cm−1 when bound with calcium, which was caused by the N−H in-plane bending vibration and C−N stretching vibration. Additionally, the peak observed at 1128.39 cm −1 became more intense and shifted to a lower wavenumber at 1114.68 cm−1, and the absorption peak of PtNH2 occurred when the SPH bound with Ca2+ to form the C−O−Ca.40 The combination between shared electron pairs of nitrogen and metal ions caused the C−N bond dipole to increase, and then the absorption was enhanced. Besides, the absorption bands of an amide IV band at 633.24 cm−1 caused by the completely in-plane vibration of the OC−N bond was moved to a lower wavenumber of 619.77 cm−1, and the strength increased. A reasonable explanation was that the electron cloud density around -CO in the OC-NH2 increased with the reduction of electron cloud density of N−H after the chelate reaction.40 The results suggest carboxyl oxygen atoms and amino nitrogen atoms as interaction sites play a primarily role in the chelate reaction of SPH and calcium ions. Changes of FTIR spectrum (Figure 7) were due to the variations of the characteristic absorption peak of the amides and carboxylates in SPH and then reflected the interaction of metal ions with organic ligand groups in the peptides.

Figure 7. Fourier transform infrared (FTIR) spectra of SPH and SPH-calcium chelate in the regions from 4,000 to 400 cm−1.

changes of the -NH stretching vibration were observed, the red shift from 3410.21 to 3398.41 cm−1 in the amide-A band indicated that the -NH2 and -NH groups participated in the chelating reaction and that the Ca−N bonds replaced the N−OH (hydrogen bonds).39 The absorption band of SPH at 1654.36 cm−1 was an amide I band possessing -CO stretching vibrations. It appeared as two peaks (1654.62 and 1647.93 cm−1) when combined with calcium, indicating that the carboxylic group involved in the covalent binding reaction with the metal ions.10 Moreover, the wavenumber at 1457.81 cm−1 for the symmetric vibration of the -COO- carboxylate group moved to a lower frequency (1437.75 cm−1) in the spectra of the chelate, and the −COOH probably turned into −COO-Ca through covalent combination with calcium ion.10 Furthermore, the 1340.14 cm−1 9710


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Figure 9. AFM topographic images of SPH (A) and the SPH-calcium chelate complex (B). Extracting representative portions from the visualized areas in A and B are shown in C and D, respectively.

Size Distribution. Particle size, depending on the volume of the particles to determine the particle radius by dynamic light scattering and Brownian motion, is one of the important parameters to measure physical properties of composites. The typical particle size distributions of the nanoparticles in SPH and the SPH-calcium chelate were shown in Figure 8. The radii of SPH and the SPH-Ca chelate were 44.70 ± 0.63 nm and 68.16 ± 0.50 nm, respectively. The particle size of the SPH-Ca chelate significantly increased when compared with the SPH, which illustrated that the chelate reaction between calcium ions and SPH not only existed in intramolecular interactions, but also existed in intermolecular interactions.13 Therefore, the interactions led to more concentrated distribution and stronger absorption strength of particles as shown by the red dotted line in Figure 8 and also indicated that folding, aggregation, and more compact structure between SPH and calcium ions occurred during the chelation procedure. Additionally, it agreed with the results gained from the fluorescence spectrometer, implying that

the presence of calcium ions could cause peptides to aggregate and fold, and further led to the formation of the SPH-calcium chelate. Beyond all doubt, the particle size distribution proved directly that the SPH-calcium chelate belonged to a compact nanocomposite. Atomic Force Microscopy. Atomic force microscopy (AFM) is a high-speed microscope captured three-dimensional image, through detecting an extremely weak interatomic interaction force between the sample surface and a microforce sensing element to study the surface structure and nature of the substance, and to obtain molecular morphology of nanometer resolution.41−43 Only water instead of the sample was used as a control experiment. Considering that the control background using water instead of the sample was only a blank image, it was not collected. Nonetheless, the images of SPH and the SPH-Ca chelate could obviously show the material morphology (Figure 9). As shown in Figure 9A, the image of SPH demonstrated a granule-like structure, and the sizes of peptides 9711


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Figure 10. 1H nuclear magnetic resonance spectra (NMR) of SPH and SPH-calcium chelate (the red line means SPH, and the green line means SPHcalcium chelate).

was enlarged, and the relaxation time increased with the addition of calcium ions, which led to the spectrum lines being broadened. The images from 1H NMR were in good agreement with the results obtained from the spectrometer, the particle size analysis, and atomic force microscopy analysis. Calcium Releasing Percentage. SPH-Ca chelate, as a potential calcium nutritional supplement, is greatly crucial to possess good solubility in the human gastrointestinal tract environment. The calcium-releasing percentages of the SPH-Ca chelate and CaCl2 at diverse pH values are shown in Figure 11.

were intensively distributed. The sulfhydryl group, carboxyl group, and amino group in the peptide chain generated aggregation because of the intermolecular hydrogen bond in the aqueous solution, then formed granules or aggregates.44 The peptides turned to granules first and then gathered into larger clusters, or a small cluster would continue to gather into very large adsorption clusters.44 Figure 9B which presents the islandshaped structures possessing fine directionality is obviously different from Figure 9A after binding with calcium ions. This is because the effects of the intermolecular forces, the surface tension, and anisotropy in the process of aggregation formed aggregates of varying directions.44 Besides, the effects in the long chain extending direction could appear in island-like aggregate morphology. Extracting representative portions from the visualized area in Figure 9A and B are shown in Figure 9C and D, respectively. The particle sizes of SPH and the SPH-Ca chelate from AFM were 50−100 nm and 50−150 nm in length, 3.0−7.0 nm and 2.25−3.50 nm in height as well, respectively. The morphology images from AFM were consistent with the results obtained from the fluorescence spectrometer and the particle size analysis. All of the results implied that a strong interaction between SPH and calcium ions brought about the formation of more compact nanostructures. 1 H Nuclear Magnetic Resonance Spectroscopy (NMR). The distribution of the electron cloud around a hydrogen nucleus was reflected by the 1H NMR spectra, which could expose the reaction process between the SPH and calcium ions through the changes of chemical shift.38 The fine peaks decreased significantly, and the spectral lines broadened distinctly as shown in Figure 10, which illustrated that the polymerization and folding phenomenon occurred during the chelate procedure. A possible cause was that some groups were substituted or strong electronwithdrawing groups in peptides reacted with other substances in the chelating reaction process, which resulted in the changes of the effects of the hydrogen proton and caused significant changes to the peak values.40 Moreover, the molecular weight

Figure 11. Calcium-releasing percentage of SPH-calcium chelate and CaCl2 at pH values of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0.

The calcium-releasing percentage of SPH-calcium chelate was always significantly higher than that of CaCl2 at all of the tested pHs. Notably, the calcium-releasing percentage of the SPH-calcium chelate maintained a relatively stable value of about 94% in the pH that ranged from 2.0 to 8.0. Nevertheless, that of CaCl2 presented an obviously downward trend from 86.85% at an initial pH value of 2.0 to 75.95% at the eventual pH value of 8.0. 9712


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Thus, it could be seen that the calcium-releasing percentage of the CaCl2 was higher in acidic conditions, i.e., calcium ions were in the dissolved state in this solution, whereas that of CaCl2 decreased gradually with the increase of pH value. Especially in the neutral to slightly alkaline condition, the calcium ions and −OH formed precipitates and resulted in the decreased percentage, which were not good for uptake and transportation in the body. It is well-known that the pH value in the human intestinal tract is higher than 7.0, approximately pH 7.2. SPH-calcium chelates with high solubility still possess significantly good bioavailability in a weakly basic environment of pH 7.2 in the gastrointestinal tract, which means that SPH-calcium chelate can be effectively absorbed and utilized through the intestinal epithelial cells.45 Therefore, the findings in our study have demonstrated that it is viable to produce a natural peptidecalcium chelate as a functionally nutraceutical supplement.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-591-22866375. Fax: +86-591-22866278. E-mail: [email protected]. Funding

This work was supported by Natural Science Foundation of China (No. 31571779), High & New Project of Fujian Marine Fisheries Department, China (No. [2015]20), the National Marine Public Welfare Projects (No. 201305022), the National Marine Biological Germplasm Resources Construction of Marine Drug Sources (No. 12PYY001SF08), and Fujian S&T Project of Fujian Provincial Science & Technology Hall (No. 2014N3005). Notes

The authors declare no competing financial interest. § J.L. and X.C. are co-first authors.

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ABBREVIATIONS USED SP, Schizochytrium sp. protein; SPH, Schizochytrium sp. protein hydrolysate; CPPs, the casein-ophosphopeptides; DH, the degree of hydrolysis; HPLC, high performance liquid chromatography; UV, ultraviolet spectroscopy; UV−vis, ultraviolet−visible spectrometry; FTIR, Fourier transform infrared spectroscopy; AFM, atomic force microscopy; 1H NMR, 1H nuclear magnetic resonance spectroscopy; D2O, deuterium oxide

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Preparation and Evaluation of the Chelating Nanocomposite

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