3.1.1 Optimization results of chelating process parameters:
3.1.1.1 Influence of the mass ratio of Nannochloropsis-metal ions :
It can be seen from Fig. 1.1(a) that the mass ratio of peptide to metal ion has a greater impact on the Effect of the chelation reaction. As the ratio increases, the chelation rate gradually increases. The chelation rate reaches its peak at a mass ratio of 9:1. Among them, the Calcium chelation rate is 51.01%, chelation activity is 7.39%, and the chelation yield is 58.76%, chelation rate increases when the mass ratio is greater than 9:1 during the reaction. Still, chelation activity decreased to 7.39%- 5.59% and the chelation yield increased to 9:1, but beyond this level, the chelation yield decreased to 46.98 (P < 0.05). (W. Wu et al., 2019) reported similar results from pig bone collagen. It is notable during the experiment at the lower level of a mass ratio of less than 9:1, peptide concentration and volume of the sample solution was constant, the concentration of metal ions was too large, the ring structure was unstable during the compounding process, and the stability of the overall complex is reduced (Pedersen, 1967). The peptide metal mass ratio gradually increases, the probability of metal ions binding increases, so the chelation yield and activity will increase; if the mass ratio exceeds the optimal value, the metal ion concentration will be low, and the probability of binding to peptide residues will decrease, and the chelation yield and activity will decrease. According to the explanation, the mass ratio of 9:1 is the optimum ratio for chelation.
3.1.1.2 Effect of Ultrasound time:
The chelation rate, activity and yield initially increased by the increase till 15mins upto 50.3%, 7.36% and 58.89% Fig. 1.1(b). After 15mins, the chelation rate, yield and activity decreased. This trend is because the energy that peptides and ions receive in the aqueous solution is low, and the reaction time is short. The chelation was not completed, whereas the more extended time with ultrasonic waves caused peptides to hydrolyze under ultrasonic cavitation and shearing (Kadam, Tiwari, Álvarez, & O'Donnell, 2015). Amino acid residues were dissociated from peptide molecules into free amino acids. The complexes formed by amino acids and metals were easily decomposed, so the metal ions were taken away, and chelation rate yield and activity were reduced. Han et al. (2015) concluded that Ca-chelated peptides' chelation rate was 67.24% at 50 mins and similar trend from Ossein peptides.
3.1.1.3 Effect of pH:
It can be seen from Fig. 1.1 (c) that the chelation rate, yield and activity increased with pH increase first and then decreased; at pH 9, the chelation rate was the highest, the Calcium chelation rate, yield and activity were 60.19%, 59.05% and 8.77% respectively(P < 0.05). This can be because of the higher concentration of H+. The electron-donating groups of the polypeptide were combined with a large number of hydrogen ions, which makes the chelation rate of divalent metal ions low. When the pH of the polypeptide solution increased from 9.0, the chelation rate, activity and yield began to decrease; at this time, there were more OH− ions in the solution, and the metal ions were hydrolyzed under alkaline conditions to form hydroxides, which cannot be used by the polypeptides (X. Wang et al., 2020). In the separation and purification process, the dissolution of metal ions and the chelation rate were greatly reduced. H. Zhang et al. (2021) reported a similar trend at pH 7 from Calcium chelated peptides of cattle bone collagen with a chelation capacity of 42.70 ± 1.09 µg/mg.
3.1.1.4 Effect of temperature:
According to the trend in Fig. 1.1(d), by increasing temperature, the chelation rate of the reaction also increased first and later decreased. The peak value occurs at a temperature of 40°, the Calcium chelation rate was 60.98 ± 0.011%, chelation activity was 8.77 ± 0.031%, and the chelation yield was 59.05 ± 0.0112. It can be explained as, lower temperature can cause lower reaction rate; and increase in temperature lead to high reaction rate till the point when the temperature was the highest resulted in destroyed cyclic structure of chelate lead to unstable reaction system. The peptide is incapacitated under the action of heat (J. Wang et al., 2020).
3.1.5 Effect of Ultrasonic power:
Figure 1.1(e) shows the trend of different ultrasonic power (0W, 150W, 300W, 500W, 1000W, 1500W) on the chelation rate, yield and activity. The optimum ultrasonic power used was 1000W with the highest chelation rate of Ca 61.89 ± 0.0091%, chelation activity 9.08 ± 0.0101% and chelation yield was 65.57 ± 0.0103%. The chelation rate varied with the increase in ultrasonic power because the ultrasonic power directly affects the extension of the polypeptide's spatial structure and the binding of metal ions. If the power is low, the polypeptide binding site cannot be fully exposed, the chelation rate is low. When the power is high, the shearing action of the ultrasound causing the chelated substance to split again, the stability at this time is relatively poor resulting in lower chelation rate.
3.2 Stability of chelated peptides:
Figure 1.2(a) shows the stability in gastrointestinal digestion. The control stability was 100%, but digestion in the small intestine with pepsin affected the chelates, Calcium retention rate was 81.93% after 30mins of digestion. Chelates were affected in acidic conditions under lower pH, resulting in instability and changing into ionic form. (Pei et al., 2022) also reported that peptides with lower molecular weight have lower stability to hydrolysis in gastric proteases. The second digestion was done by adding trypsin with a prolonged time, and with trypsin addition, the calcium detention rate was significantly reduced. It was because of acidic conditions, the chelate structure changed significantly to bind Calcium ions; trypsin results in the degradation of peptides and a decrease in chelating activity. The Stability of Calcium chelated peptides is important to study because during gastrointestinal digestion conditions, Calcium ions tend to react with phytic acid or oxalic acid in the stomach resulting in insoluble precipitates or the formation of Ca(OH)2 in the small intestine, resulting of lower bioavailability of Calcium (X. Yang et al., 2021). The present study indicated that the Calcium chelates prepared were stable in gastrointestinal digestion conditions that can naturally improve Calcium absorption. At phosphate buffer lower concentration (≤ 10mmol/L), the chelates were stable, and the Calcium retention rate was more than 75%.
Figure 1.2 (c) shows the pH stability of chelates at different pH levels. Initially, at lower pH 3 at 46.23 ± 0.0231%, the stability was lower, as explained in gastrointestinal digestion. By the time the pH increased, the stability of chelated peptides had increased to 98.44%± 0.00145 at pH 9 (P < 0.005). It has been explained that an increase in the pH H + concentration under acidic conditions could compete with calcium ions for active binding groups, enhancing chelates' dissociation. These results are similar to the lower chelation rate in Fig (a) in gastrointestinal digestion. Fig (e) shows the Effect of temperature on chelates. Initially, the stability decreased, with the increase in temperature, it remained unchanged significantly (P < 0.005). It can be concluded that chelates are resistant to thermal processing. Similar results were presented by (H. Zhang et al., 2021) (X. Yang et al., 2021).
3.3 Molecular weight distribution analysis of chelates and peptides:
Researchers have reported that molecular weight distribution correlated to the Calcium binding capacity of peptides(Kong et al., 2023; X. Sun et al., 2020). Results show that the profile of different molecular weight peptides and chelated peptides were divided into 4 groups (< 200kDa, 200-1kDa, 1-3kDa, and > 3kDa). Figure 1.3 shows that Nannochloropsis peptide and chelate peptide-Ca are dominated by peptides below 3000 Da (78.08% and 64.89%, respectively). The content of peptides components with a molecular weight of is less than 3 kDa, in which components with a molecular weight of 200-1k Da is about 60%。The 200–1000 Da range is the main molecular weight distribution interval for both Nannochloropsis peptide and chelate peptide-Ca, accounting for 52.3% and 54.34%, respectively. These results indicate that Nannochloropsis peptides with 200–1000 Da molecular weight are better with binding metal ions and have higher chelating activity.
Many studies concluded that higher molecular weight peptides have higher chelated activity, such as peptide fragments with Calcium binding capacity having a molecular weight lower than 2000Da. A study concluded that Calcium-binding peptides had a molecular weight of 1033 Da derived from tilapia scale protein hydroslates(He et al., 2022) (D. Chen et al., 2014).
3.4 Amino acid composition analysis:
Many studies have reported that peptide amino acid composition affects chelating ability(He et al., 2022). Glutamic acid and aspartic acid are the most important in chelation, accounting for 26.2% and 14.6% in Calcium binding peptides. Leucine, isoleucine, and phenylalanine played a crucial role in the process of chelation (J. Wu, Cai, Tang, & Wang, 2019). The chelating ability of peptides was affected by acidic amino acids (Xu Wang et al., 2017).Table 1.1(a) explains the types, numbers and amounts of different amino acids in peptides and chelated peptides. Studies have reported that the amino acids responsible for chelation i.e. carboxyl group of acidic amino acids, and negative charges of hydrophobic amino acids were mainly responsible for chelation (Tavafoghi & Cerruti, 2016). Comparison of peptides and chelated peptides illustrates the relative content of hydrophobic amino acids in peptides and chelated peptides are 41.6%and 23.2%, respectively. As can be seen from Table 1.1(a), the proportions of Glu and Asp are significantly higher, 26.2% and 14.6%, respectively. Therefore, it can be assumed that the amino acids involved in the chelation reaction are mainly Glu and Asp.
Moreover, the nutritional value of peptides is directly related to the proportion of essential amino acids, which is 49.8% and 33.3% in peptides and chelated peptides, respectively.
3.5 Fourier transform infrared spectroscopy analysis of chelates and peptides:
Figure 1.4(a) shows the FTIR graphs peak of Calcium peptides with enzymatically hydrolyzed peptides. The highest frequency absorption for peptides is 3,284.69cm− 1 can be characterized as the stretching of N-H in the Nannochloropsis peptides. Hence, the chelated peptides showed Ca is 3,281.78cm− 1, indicating that the N-H cloud density got more potent because of the dipole and induction effect as N-H indicates the chelate formation (Wenjuan Qu et al., 2022). Similar findings were reported by W. Wu et al. (2019) using Ca-chelated peptides, and the highest frequency of chelated peptide was reported at 3409 cm− 1. The amide I band in the peptides and chelates for peptides 1,644.90cm− 1 Ca 1,644.73cm− 1. Initially, in the enzymatically hydrolyzed peptides, the absorption was 1,644.90cm− 1. After chelation complex formation, it shifted to lower absorption Ca 1,644.73cm− 1. This explains how the C-O contributed to calcium chelation with covalent bonding .The ECOOH bounding with the Calcium in the chelated product (COO-) as the enzymatic peptide showed wavelength of 1590.27cm− 1 and shifted to1,567.46cm− 1.Here the chelation was done because of the free electron pair of carbonyl oxygen to chelate Calcium (L. Wang et al., 2018). Bending vibration NeH can be attributed to band 1,398.30cm− 1 to Ca 1405.87cm− 1. C-N stretching vibration was changed from 1243.78cm− 1 to 1235.94cm− 1. It can be concluded that the chelation was done by calcium ions bounded with amino nitrogen atoms and carboxyl oxygen of peptides, which can relate to the findings (Peng, Hou, Zhang, & Li, 2017). (W. Wu, Li, Hou, Zhang, & Zhao, 2017a, 2017b) reported similar findings that iron chelation was done by bonding carboxyl groups and amino nitrogen atoms with mineral ions.
3.6 UV-VIS spectroscopy of peptides and chelated peptides
Figure 1.4(b) shows the UV-vis spectroscopy analysis. The most substantial absorption peak for peptides was visible at 333nm; for Calcium chelates, it was visible at 342nm, which was constant with the characteristic peak of a peptide by the n → π∗ transition of C = O chromophore and phenylalanine in peptide bonds (Xu Wang et al., 2017). Chelates showed higher absorption intensity in the near-UV region than peptides because the transition metal ions absorb light; another reason can be the electron transition was caused by the internal transition of chelates due to the absorption of the UV region (J.-L. Chen et al., 2013). Moreover, there was a gentle stage between 350–450 nm before flattening the spectrum, which can be due to the chirality of the contained metal binding sites such as -CO-, -NH-, -NH2-, -COOH, and so on (Lefringhausen, Seiffert, Erbacher, Karst, & Müller, 2023; Tkaczyk, 2009). Similar findings were reported in many studies, such as (Luo, Yao, Soladoye, Zhang, & Fu, 2022; L. Wang et al., 2018; Zhao et al., 2014). UV-vis spectrum confirmed the production of new complexes as chelates.
3.7 Fluorescence spectroscopy of Calcium and Ca chelates
A comparison of peptides and chelates structure can be seen in Fig. 1.4(c). The calcium-chelated peptide showed a fluorescence absorption band shifted in position compared to peptides. The intensity of the chelated peptides is higher than the peptides at a wavelength of the peptides themselves because of the new structure identified by the reaction of chromophores with Calcium ions that results in the excitation of energy leading to state change. Secondly, the folding of the peptide was done by Calcium ions, which can be the reason for variation in the state of the fluorescence intensity (Zhai et al., 2023)
3.8 Zeta potential of chelates and peptides :
The Zeta potential showed that the average potential of the peptide was − 19.80 MV, and the negative value decreased to -15.97 MV after the peptide was bound to the Calcium ion. Results showed that the Nannochloropsis peptides and Calcium ions formed new compounds. The zeta potential of Peptides-Ca is lower than that of peptides, so the peptide Ca was more unstable and tended to become bigger particles by folding and aggregation than peptides. Hence, it can be concluded that the Calcium ions in the medium were surrounded by chelating sites such as amino and carboxylic groups of microalgal peptides with coordinate bonds exhibiting the neutral molecules which help in stability in the gastrointestinal tract (L. Zhang, Lin, & Wang, 2018).
3.9 TG-DSC analysis of chelates and peptides:
As shown in Fig. 1.4(d), the DSC curves of peptide and peptide -Ca appeared to be similar; there were no apparent peaks, showing no thermolysis of peptide and peptide -Ca by thermal decomposition. The TG curves of peptide showed that the increase in downsizing was shown as the green lines, depending on the TG graph's slope, which lost 25.89% weight entirely, from 0-200 ℃. However, peptide-Ca lost 14.05% weight from 0 to 200 ℃. DTG (0.4% min) is formed − 1 to 0.1 in the peptide –Ca, and DTG (0.1% min) is formed − 4 to 0.5 in the peptide. The significant curve decline in the TG slope was mainly due to the C-N bond at different positions of peptides (Shao, Wang, Zhang, Zhang, & Hao, 2022). The superficial difference in the temperature of constant weight indicated that peptide -Ca formed more stable and thermo-stable than peptide (Wenjuan Qu et al., 2022).
3.10 X-ray diffraction analysis of chelates and peptides:
The differences in both spectra show a substantially change x- diffraction pattern in chelated peptides. Figure 1.4(e) shows that the amino acid composition of Nannochloropsis peptides was not stable. Hence, Nannochloropsis peptides appeared amorphous, and spectra shows no absorption peak. Moreover, after the chelation process, the Ca-chelated peptides in X-diffraction showed clear peaks and stronger absorption. X-diffraction of chelating peptide has small end spectra, a high and sharp diffraction peaks explain the formation of good crystal. The peptides show strong and dispersed peak at 2θ = 24.56.35 which explains the amorphous structure, but after the chelation with calcium chelates, they exhibited two crystal diffraction peaks 2θ = 6.06◦ and 2θ = 17.86 with narrow bases. W. Qu et al. (2022) reported similar findings to confirm changes in the crystalline structure of peptides and chelates complex
3.11 X-Ray Photoelectron Spectroscopy Spectral analysis of chelates and peptides:
Surface chemistry was done using XPS. When exposed to X-rays, the XPS instrument measured the kinetic energy emitted from the surface elements. Previously it has been used in iron-chelated peptide characterization from Antarctic krill and Pumpkin (Cucurbita pepo L.) seeds (H. Hou et al., 2018; Lu et al., 2021). Figure 1.5 shows the content and elemental composition of both peptides and chelates. It can be seen that peptides were composed of Fe (0.29%), O (41.44%), N (10.72%), Ca (0.58%) and C (31.63%) whereas; chelates composition was Fe (1.04%), O (41.50%), N (9.20%). Ca (4.78%) and C (46.96%). The increase in calcium content in chelates upto 4.02% indicates the interaction of calcium with peptides to form chelates. The abovementioned changes were also confirmed using FTIR, UV-vis and XRD analysis.
3.12 Scanning electron microscope analysis of chelates and peptides:
The smooth structure with some particles explains the free Calcium release during the preparation of peptides during hydrolysis that is absorbed on the surface of peptides Fig. 1.6(a). The chelate surface appeared looser with spherical aggregations. The dense structure of peptides was destroyed by the chelation process by the interaction between the peptide and Calcium so there was a difference between the peptide microstructure and chelates structure(Khalil, Booles, Hafiz, & El-Bassyouni, 2018; Xu Wang et al., 2017; W. Wu et al., 2019). Some crystals were also seen on the chelates' surface, indicating that some salts were adsorbed on the surface of the peptides, as reported in some previous studies (Khalil et al., 2018; Peng et al., 2017).
3.13 AFM analysis of chelates and peptides:
The intermolecular aggregation of the chelates caused by an amino group, sulfhydryl group and carboxyl group in the peptides is clearly shown in the image as a blocky appearance of chelates (J. Lin, Cai, Tang, & Wang, 2015) (J. Lin et al., 2015; Y. Lin, Cai, Wu, Lin, & Wang, 2020). Figure 1.6(b,c) shows that chelates surface peaks are more prominent and denser than peptides. These results explain the difference in aggregation on the atomic microstructure of peptides and chelates morphology. Similar results were discussed in the study of cattle bone collagen peptides (H. Zhang et al., 2021)