Sustainable and one-pot fabrication of peptide chelated calcium from fish scale hydrolysates

Fish scales, considered as low-value by-products, contain peptides and hydroxyapatite that can be applied to produce peptide chelated calcium directly. This study developed a sustainable and one-pot fabrication method for the pep-tide-chelated calcium from fish scale hydrolysates (FSP-Ca). During pepsin hydrolysis, the releases of peptides (FSP), calcium, and phosphate from fish scales occurred simultaneously, and the chelation was also effectively performed. After a 6-h hydrolysis, the yield of FSP was 46.18%, and the dissolution rate of calcium was 49.53%. Under the optimal conditions (pH 7, chelation time of 25 min, and chelation temperature of 48 °C), a high chelation rate of 86.16% was obtained, with a calcium content of 81.8 mg/g. The results of UV absorption, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) confirmed the successful chelation between FSP and calcium derived from fish scales. The –NH 2 , –COO – , N–H, C=O, C–H, and –OH groups in FSP participated in the formation of FSP-Ca.


Introduction
Calcium is a vital component of human bones and teeth.It plays a crucial role in various physiological activities, such as intracellular metabolism, cardiac function, muscle contraction, and bone growth [1,2].For example, a deficiency in calcium can lead to several disorders, including hypertension, rickets, and osteoporosis [3,4].The recommended intake of dietary calcium for individuals over 19 years of age varies from 1000 to 1300 mg, depending on the reference guidelines [5].However, dietary calcium intake is notably low in many Asian countries (< 500 mg/day), and similarly inadequate intake levels (400-700 mg/day) are observed in parts of Africa and South America [6].Consequently, developing effective calcium supplements has become a significant research focus to address widespread calcium deficiencies.Nowadays, the ionic calcium supplement (e.g., calcium gluconate, calcium carbonate and calcium lactate) is the main calcium supplement on the market.However, these calcium supplements are easy to form calcium precipitates in the gastrointestinal tract, thus resulting in a low bioavailability [7].Contrastively, the peptide-calcium chelates can remain soluble during digestion, in which the chelated calcium can be absorbed through the transportation channel of small peptides with high bioavailability and fast absorption [8,9].
The peptide chelated calcium belongs to cyclic complexes of peptides bound to calcium ions in the form of ligand and ionic bonds [10].Currently, the processes of preparing peptide chelated calcium can be divided into two steps, that is, first preparing the peptide by enzymatic hydrolysis, and then mixing the peptide with the inorganic calcium source (e.g., calcium chloride) to complete the production of chelates [7,11,12].In addition, the phosphorylation modification of peptides before chelation has been considered as an effective approach to improve calcium-binding capacity of peptide, which is mainly attributed to the electrostatic interaction between calcium ions and the negatively charged phosphate groups [13][14][15].Overall, the present preparation of peptide-chelated calcium is still complicated and time-consuming.
Fish production is a growing industry and a huge amount of fish processing byproducts (e.g., skin, scale and bone), accounting for 50-70% of the whole fish, is produced, whose efficient utilization could bring high commercial value and reduce potentially environmental pollution [16][17][18].For example, fish scales are generally regarded as wastes from aquaculture and food processing enterprises during the production of fish canning, filleting, salting and smoking.Actually, fish scales contain 41-45% of organic components (e.g., collagen, fat, and lecithin), 38-46% of inorganic components (e.g., hydroxyapatite and calcium phosphate) and trace elements (e.g., calcium and magnesium) [19][20][21].Especially, fish scales mainly composed of type I collagen, calcium and hydroxyapatite, which can be used as integrated sources of peptide, calcium and phosphate for preparing peptide chelated calcium [22,23].However, few studies have highlighted fish scale as a source of calcium and peptides.
Notably, fish scales are the mineralized plates covering the dermis layer of the epidermis as a protective shield of skin [20].In order to achieve the release of calcium ions and phosphate in fish scales, the treatment needs to be carried out under acidic conditions.However, some previous studies have shown that the conditions for phosphorylation of peptides and chelation of calcium are closer to neutral [14,24,25].Generally, sodium hydroxide is widely used as a common pH regulator.However, its use can not only introduce extra sodium salts, but also result in the binding of hydroxide ions to calcium to form a precipitate and hinder the chelation reaction [26].Therefore, developing a facile and efficient preparation process of peptide chelated calcium is of great significance.Theoretically, simultaneous releases of peptides, phosphate and calcium ions from fish scales could be achieved during enzymatic hydrolysis under acidic conditions.However, there is little information available regarding how to use the main components of fish scale (e.g., collagen, hydroxyapatite, and calcium) to produce peptide chelated calcium, while avoiding the use of sodium ions.
In this study, both calcium and peptides could be extracted from grass carp scales by pepsin hydrolysis and then they were applied to prepare peptide-calcium chelate using a facile one-pot strategy.This strategy can realize the rational utilization of fish scale components and reduce production costs.The release process of peptides, calcium ions and phosphate from fish scales under acidic conditions was investigated.Subsequently, the phosphorylation of peptides and their chelation with calcium were simultaneously completed after the innovative use of arginine to regulate pH.In the process of chelating peptides with calcium ions, arginine can solve the issue that hydroxide ions tend to combine with calcium to form precipitation in the traditional process of pH adjustment using alkali and prevent calcium ions from participating in chelation, while avoiding the introduction of additional sodium salts.Notably, arginine can also be used as an amino acid component to assist chelate reaction.The optimal chelation conditions were determined by response surface methodology (RSM).The amino acid composition and characterization of peptide chelated calcium were determined.

Materials
Fresh grass carp scales were collected from a local Yonghui Supermarket (Beibei District, Chongqing, China).Immediately after washing with tap water to remove impurities and blood, the scales were dried at 60 °C for 5 h in a drying oven and then stored at room temperature for further use.Pepsin (1:10,000 units), Coomassie brilliant blue R-250 and bovine serum albumin (BSA) were provided by Bio Basic (USA).Arginine and trichloroacetic acid (TCA) were provided by Aladdin Reagent Co., Ltd.(Shanghai, China), and triethanolamine, chromium black T, disodium glycolate tetraacetate were provided by Chuandong Chemical Co., Ltd.(Chongqing, China).Other chemicals and reagents used were of analytical grade unless stated otherwise.

Preparation of fish scale peptides (FSP)
Briefly, the pretreated fish scales were mixed with an acidic solution (pH 2) at a solid/liquid ratio of 1:20 (w/v), followed by heating at 85 ℃ for 1 h to induce scale swelling and loosening.Afterwards, the pepsin (240 U/g) was added and maintained at an optimal condition (pH 2, 37 ℃) to complete the enzymolysis.After various enzymolysis times (i.e., 1, 2, 3, 4, 5, 6 and 7 h), the resulting sample was immediately placed in a boiling water bath for 15 min to inactivate pepsin.The enzymatic hydrolysate was centrifuged at 8500 rpm for 20 min, then the obtained supernatant was freeze-dried using a vacuum freeze dryer (FD-1-50, Beijing Bo Yikang Experimental Instrument Co., Ltd., China).Finally, the dried FSP was available for further use.The yield of FSP was determined by the traditional biuret method [27].

Determination of degree of hydrolysis (DH)
The DH was measured according to the o-phthalic aldehyde (OPA) method with appropriate modifications [28].Briefly, 100 µL of the diluted hydrolysates was mixed with 2 mL of OPA solution.After 2 min incubation at room temperature in dark, the absorbance was recorded at 340 nm using a synergy HTX multi-mode reader (BioTek instruments, Winooski, Vermont, USA).L-leucine was applied to create a standard curve and then used to calculate the content of free amino groups.Total acid hydrolysis was performed by adding 6 mol/L HCl to the scales and stirred at 110 °C for 22 h prior to analysis.The absorbance was read at 340 nm using microplate reader (Multiskan GO; Thermo Fisher Scientific).The DH (%) of fish scales was calculated using the following equation: (1) where [NH 2 ] represents the free amino acid concentration in the enzymatic digestion solution (mol/L), V 1 is the volume of enzymatic digestion solution (L), m 1 is the mass of fish scales used for enzymatic digestion (g).
[NH 2 ] 2 represents the free amino acid concentration in the original hydrolysate obtained after acid hydrolysis (mol/L), V 2 is the volume of acid hydrolysate (L), m 2 is the mass of fish scales used for acid hydrolysis (g).

Determination of degree of phosphorylation
The molybdenum blue colourimetric method was used to determine the phosphorus content of the enzymatic hydrolysate, according to the procedure described in Chinese National Standard GB 5009.87-2016[29].After digestion, phosphorus can be reacted stoichiometrically with ammonium molybdate to form ammonium phosphomolybdate in the presence of a reducing agent (hydroquinone, sodium sulfite or stannous chloride and hydrazine sulfate).The absorbance at 660 nm was recorded to calculate the concentration of phosphorus.

Determination of dissolution rate of calcium
The dissolution rate of calcium in the resulting enzymatic hydrolysate was measured using ethylenediaminetetraacetic acid (EDTA) complexion titration method [30].
The standard curve was plotted with the EDTA titration volume (x, mL) as the horizontal coordinate and the calcium ion concentration (y, mg/mL) as the vertical coordinate.The calcium ion concentration (y, mg/mL) in the enzymatic solution was calculated from the standard curve (y = 0.0944x + 0.0608, R 2 = 0.9997), and the calcium dissolution rate was calculated using the following equation: where C is the calcium ion concentration in the enzymatic digestion solution (mg/mL), V is the volume of enzymatic digestion solution (mL), f is the dilution multiple, m is the mass of the fish scale (mg), and X is the mass fraction of calcium in the fish scale (%).

Preparation of peptide-calcium chelate from fish scale (FSP-Ca)
The chelation reaction between calcium and FSP was carried out in a water bath under different chelation conditions (i.e., pH: Following the chelation reaction, the product was further (2) purified by adding 7 times the volume of anhydrous ethanol and placed at 4 °C for 6 h.After being centrifuged (10,000 r/min, 15 min), the precipitate was collected and freeze-dried for 48 h.The obtained peptide-calcium chelate powder from fish scale was available.Based on single factor test results, a Box-Behnken design (three-factorthree-level) was employed using Design-Expert 13.0 software to optimize the conditions.The chelation rate of calcium was calculated using the following equation: where C is the total calcium ions in solution (g), m is the free calcium content in supernatant (g).

Amino acid composition
The amino acid composition of the lyophilized FSP and FSP-Ca was determined according to National Standard of the People's Republic of China (GB 5009.124-2016,Determination of calcium in foods), using an L-8900 Automatic Amino Acid Analyzer (Hitachi High-Tech Science Co., Japan).

Ultraviolet absorption spectroscopy
The lyophilized FSP and FSP-Ca were dissolved in distilled water at a concentration of 1 mg/mL.Then, the spectra of the solution were recorded with an ultravioletvisible spectrophotometer (UV-6100, Metash, Shanghai, China) in a wavelength range of 190-400 nm using distilled water as a reference.

Fourier transform infrared spectroscopy (FTIR)
FTIR spectra of the lyophilized FSP and FSP-Ca were recorded in the range of 4000-600 cm −1 at a resolution of 4 cm −1 using a FTIR spectrometer (Thermo Nicolet iS10, Thermo Fisher Scientific, USA).

X-ray diffraction (XRD)
XRD measurements of the lyophilized FSP and FSP-Ca were carried out using an X-ray diffractometer (X'Pert3 Powder, Malvern Panalytical, Netherlands) with a Ni-filtered Cu-Kα radiation (λ = 0.154 nm) operated at 40 kV and 40 mA.The scan angle (2θ) ranged from 10° to 80° at a scanning rate of 2°/min and scanning step width of 0.02°.

Scanning electron microscopy (SEM)
The morphologies of the lyophilized FSP and FSP-Ca were observed using a scanning electron microscope (Phenom Prox, Phenom-World, Netherlands) under an (3) accelerating voltage of 10 kV.Prior to observation, the samples were uniformly sprayed and sputter-coated with gold.

Statistical analysis
All experiments were performed in at least triplicate, and the data were expressed as the mean ± standard deviation (SD).Statistical analysis was performed using SPSS (IBM Corporation, version 22.0).Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test at a fixed significance level (P < 0.05).

Effect of enzymatic hydrolysis time
Effects of pepsin hydrolysis time on production of FSP was investigated.The yield of FSP and dissolution rate of calcium were used as the main indicators, while the DH and release of phosphate served as the auxiliary indicators, and the results are displayed in Fig. 1A and B, respectively.As can be seen from this figure, the DH of fish scale displayed an obvious increase with the increase of enzymatic time (1-6 h) and then remained stable after 7 h (DH of 5.5%).At the initial stage of enzymatic digestion, the substrate is sufficient accompanied with more enzyme active sites, thus the DH increased rapidly in the initial stage (1-3 h).Thereafter, as the time prolonged, the proteins in the fish scales were gradually consumed with the accumulation of the hydrolyzed products, while the corresponding decrease in enzyme activity led to the inhibition of enzymatic digestion, thus slowing down the increase in DH [31].Additionally, the release of phosphate also gradually increased with increasing enzymatic digestion time, reaching a maximum value (20 mg/g) at 7 h.Phosphorylation is an enzyme-catalyzed esterification reaction in this experiment, where FSP can bind to phosphate groups in hydroxyapatite derived from fish scales under acidic enzymatic hydrolysis conditions.This process could endow FSP with a large number of electronegative ions, thereby improving its ability to chelate calcium ions [14].As the extension of enzymatic digestion time, the yield of FSP and the dissolution rate of calcium also increased gradually.Finally, the dynamic equilibrium was achieved at 6 h, showing a FSP yield of 46.18% and dissolution rate of calcium of 49.53%.Thus, the optimum value of enzymatic digestion time was 6 h.

Single factor test for the preparation of FSP-Ca
The effects of pH, temperature and time on chelation rate for the preparation of FSP-Ca are displayed in Fig. 2. In this experiment, arginine (10%, w/v) as a type of alkaline amino acid was used to replace sodium hydroxide as pH regulators.As can be seen from Fig. 2A, the chelation rate increased significantly when the pH value increased from 4 to 7. A gradual increase in pH can reduce the competitive effect, in which hydrogen ions compete with calcium ions to bind negatively charged amino acid residues in FSP.Additionally, at pH 4, 5, 6 and 7, the degree of phosphorylation of FSP also increased significantly with increasing pH (namely 0.22, 0.25, 0.31 and 0.42 g/100 g, respectively).This phenomenon also suggested that the change in pH through arginine modulation method could induce the phosphorylation of peptide, which was more conducive to the chelating reaction of peptide and calcium ion.At pH 9, the chelation rate showed a slight decrease due to the formation of calcium hydroxide precipitates [24].Therefore, the optimum pH for chelation was 7.0.The effect of temperature on the chelation rate is shown in Fig. 2B.Although the effect of temperature on chelation rate was not significant, the chelation rate increased gradually with increasing temperature and reached a maximum value at 45 °C, and then decreased with the gradual increase in temperature (45-75 °C).An appropriate temperature would accelerate the molecular motion to promote chelation reaction, while too high temperature could induce conformational changes in peptides and dissociation of chelates, thus decreasing the chelation rate.Thus, the optimum temperature for the chelation reaction was 45 °C.As depicted in Fig. 2C, there was no significant difference in chelation rate as time increased from 15 to 90 min, and the maximum value was obtained after chelating for 30 min.It indicated that the chelation reaction between FSP and calcium was rapid, which was also confirmed in other published studies [32,33].Based on the above results, the optimal chelating parameters were considered for the following RSM experiments: pH of 6-8, temperature of 40-50 °C, and time of 20-40 min.

Optimization of chelation conditions by RSM
Based on the results of single factor tests, the range and center point values for the three independent variables (i.e., pH, time and temperature) are presented in Table 1.Then, the chelation conditions under different pH, time and temperature were further optimized for the preparation of FSP-Ca using a three-factor three-level BBD with 17 runs, and the results of BBD were also displayed in Table 1.The following second-order polynomial regression equation showed the chelation rate (Y) as a function of pH (A), time (B), and temperature (C), by applying multiple regression analyses to experimental data: ANOVA is applied to evaluate the adequacy and significance of the model, and the results are summarized in Table 2.A very low P-value of the proposed model (P < 0.0001) with the calculated F-value (381.46) confirmed that the model was extremely significant and credible.Additionally, lack of fit (0.4469) was not significantly relative to the pure error, and the value of R 2 (0.9980) was satisfactory, which implied that this model fitted the experimental data well with the actual test and described the relation between response and factors reasonably [24,34].Moreover, the results showed that two linear terms (A and C) and three quadratic terms (A 2 , B 2 and C 2 ) were significant (P < 0.05), while one linear term (B) and three interaction terms (AB, AC and BC) were not significant (P > 0.05).
Generally, the 3D response surface plots and contour plots generated from regression equation can be used to predict the relationships between the independent and dependent variables.Figure 3 displays the central optimum point of the 3D response surface plots and contour plots for predicting maximum chelation rate.Especially, the steeper the 3D response surface corresponding to each factor, the greater the effect of that factor on the response value.Thus, the steepest slope of the surface belonging to pH was the most significant among the three factors, which was consistent with the results of ANOVA.
As shown in Fig. 3A and D, the chelation rate raised at the beginning, and plateaued with the increase of pH (6)(7)(8) and chelation time (20-40 min).Similarly, with the increase of pH (6)(7)(8) and chelation temperature (40-50 ℃), the same changing trends in chelation rates were also observed (Fig. 3B and E).However, as observed in Fig. 3C and F    recommended as: pH 7.11, chelation time of 25 min, and chelation temperature of 48 °C, in which the highest chelation rate was predicted to be 86.61%.Under this condition, the validity of the proposed model was further verified by a validation test.Considering the convenience in actual operation, the predicted values were adjusted: pH 7; chelation time of 25 min; and chelation temperature of 48 °C.Under these conditions, the actual chelation rate was 86.16 ± 0.30%, which was similar to the predicted value without significant difference.The calcium content of FSP-Ca was 81.8 mg/g.

Amino acid composition analysis
The amino acid compositions of FSP and FSP-Ca are summarized in Table 3.The results suggested that FSP was rich in glycine (25.63%), arginine (9.16%), alanine (11.02%) and glutamic acid (11.32%).After chelation with calcium, there was little difference in the variation of each amino acid content in FSP-Ca.Notably, the content of hydrophilic amino acids increased after chelation, indicating that hydrophilic amino acids were beneficial to the formation of chelates [38].Generally, the ability of peptides to chelate with metal ions is closely related to the type of amino acids, among which aspartic acid, alanine, glutamic acid and arginine have been shown to provide binding sites due to the presence of free carboxyl, amino and imidazole groups, whose content has a positive correlation with the binding ability of peptides [35][36][37].Besides, glycine was also identified as one of the major amino acids that can chelate with calcium [25].Additionally, the content of arginine changed obviously from 9.16% in FSP to 14.09% in FSP-Ca, which was mainly due to the addition of arginine during chelation for pH regulation.It was calculated that 76.76% of the added arginine was used to participate in the chelation reaction, while only 0.038% was involved in adjusting pH of the enzymatic solution.

UV absorption spectroscopy analysis
The difference between FSP and FSP-Ca can be reflected by the intensity and dislocation changes of UV absorption spectra.As shown in Fig. 4A, FSP displayed the maximum absorption peak at 223 nm and a weak absorption peak at 275 nm, respectively attributing to the n → π* transition of carbonyl, carboxyl, amide bonds and aromatic amino acid residues in FSP [24,39].However, the maximum absorption peak showed a blue shift and transfer from 223 to 205 nm after chelating with calcium.In addition, the spectrum of FSP-Ca was flatter than FSP in the range of 250-300 nm, and the weak absorption peak at 275 nm disappeared.This might be also due to the interaction that the chirality of the chromospheres and auxochromes involving C = O, COOH, -OH and -NH 2 changed when FSP combined with calcium ions [39].
During chelation, the energy required for the electron transition after the ligand binds to the metal ion is different from the energy required for the free ligand itself, thus leading to the difference in UV absorption spectra [40].A similar phenomenon was also found in the UV spectra analysis of cucumber seed peptide and peptidecalcium chelate [39].Thus, the UV absorption analysis confirmed the successful chelation between FSP and calcium to form FSP-Ca.

FTIR analysis
When calcium ions effectively bind to the organic functional groups in FSP, the characteristic absorption peaks in FTIR spectra could be obviously changed due to the vibration of the coordinate bonds.Confirmatively, FTIR spectra of FSP and FSP-Ca are depicted in Fig. 4B.In the spectrum of FSP, the absorption peaks at 3253, 1628, and 1412 cm −1 were attributed to the stretching vibration of -NH 2 , C=O, and -COO -, respectively.After being chelated with calcium ions, the obtained FSP-Ca showed obvious difference in comparison with the spectrum of FSP.Especially, the absorption peak for -NH 2 shifted from 3253 to 3287 cm −1 in FSP-Ca, attributing to the transfer of electron pairs from nitrogen atoms to calcium, forming N-Ca instead of the N-H bond [41].The amide I band observed at 1628 cm −1 in FSP shifted to 1641 cm −1 in FSP-Ca, indicating the participation of carboxylic group in chelating and the formation of α-helix structure in FSP-Ca [24].Furthermore, the peak for -COO -also shifted from 1412 to 1404 cm −1 in FSP-Ca, indicating that the carboxylate group of FSP was effectively bound to calcium ions [2,41].In the fingerprint region, the peak changed from 1080 to 1036 cm -1 , indicating that C-H and -OH bonds also participated in the chelating reaction [25].The small peak at 938 cm -1 represented the inplane bending vibration of the O-H bond in the carboxyl group, but it disappeared when combined with calcium ions [37].Based on the abovementioned results, the -NH 2 , -COO -, N-H, C=O, C-H, and -OH groups in FSP might participate in the formation of FSP-Ca.

XRD analysis
The XRD patterns of FSP and FSP-Ca are shown in Fig. 4C.It can be seen from the figure that FSP showed a relatively broad and weak diffraction peak at 2θ = 23.37°,implying a random amorphous structure.After chelating with calcium ions, XRD patterns of the obtained FSP-Ca significantly differed and appeared some new peaks at 2θ = 13.25°,33.79°, 52.24°, 54.91° and 62.89°.The chelating of FSP and calcium generated new interaction forces of the new ordered crystal structure, thus displaying new diffraction peaks.The presence of calcium ions could induce the folding of peptides and the increase in crystallinity of FSP, promoting the formation of the ordered crystal structure of FSP-Ca [25].Several similar studies about peptides-calcium chelates also found the formation of new crystalline structure [42,43].

SEM analysis
SEM images of FSP and FSP-Ca are shown in Fig. 4D.As can be clearly observed from the figure, the microstructure of FSP and FSP-Ca showed great difference after chelating process.Obviously, the surface of FSP was relatively dense and smooth with a subtle furrow, while the surface structure of FSP-Ca became rough and loose with aggregation of dense crystal particles, which has also been observed in other works about peptide-calcium chelates [14,30].This phenomenon was caused by a combination of coordination binding of FSP to calcium ions and the trapping of calcium by the bridging role of FSP accompanied by its structural folding and aggregation [35,44].Overall, the presence of more crystals on the surface of FSP-Ca confirms the binding of calcium ions to FSP and the formation of chelates.

Conclusion
The present study facilely fabricated the peptide chelated calcium from grass carp scales through a sustainable and one-pot method.The DH, peptide yield and dissolution rate of calcium and phosphate in fish scales showed an increasing trend with the increase of pepsin hydrolysis time, having an optimal hydrolysis time of 6 h.Through optimized chelation experiments by RSM, the following conditions (i.e., pH 7, chelation time of 25 min, and chelation temperature of 48 °C) were optimal and displayed a high chelation rate (86.16%) and calcium content (81.8 mg/g).The results of UV absorption, FTIR, XRD and SEM all showed that FSP successfully chelated with calcium ions, and the structure of FSP-Ca was different from that of FSP.The carboxyl and amino groups of FSP and hydrophilic amino acid in FSP participated in the chelation with calcium as the primary binding sites to form FSP-Ca.This study provides an innovative strategy to prepare fish scale peptide chelated calcium as calcium supplement.Further studies should be warranted to analyze the influences of calcium chelation reaction on the bioactivities of FSP-Ca and the pathway of calcium transport and absorption mechanism in vivo.

Fig. 1
Fig. 1 Effect of enzymatic hydrolysis time on the yield of FSP and calcium dissolution rate A, and DH and release of phosphate B

Fig. 2
Fig. 2 Effect of reaction pH (A, reacted at 45 °Cfor 45 min), temperature (B, reacted at pH 6 for 45 min) and time (C, reacted at pH 6 and 45 °C) on chelation rate , the effect of chelation temperature (40-50 ℃) and chelation time (20-40 min) on chelation rate was relatively weak.Based on the results of response surface model, the optimum parameters of the test variables were

Fig. 3
Fig. 3 Response surface plots A, B and C and contour plots D, E and F showing the interactive effects of pH, temperature and time on chelation rate

Fig. 4
Fig. 4 UV A, FTIR B, XRD C spectra and SEM images D of FSP and FSP-Ca

Table 1
Experimental data for chelation rate from the Box-Behnken design by RSM

Table 3
Amino acid composition of FSP and FSP-Ca