Discovery of calcium-binding peptides derived from defatted lemon basil seeds with enhanced calcium uptake in human intestinal epithelial cells, Caco-2

Abstract

It is anticipated that calcium-chelating peptides may serve to enhance the absorption of calcium. This research examined defatted lemon basil seeds (DLBS) which had been treated with Alcalase under optimized parameters for the degree of hydrolysis for proteolysis, discovering that the activity for calcium-binding in a competitive condition with phosphate ion was 60.39 ± 1.545%. The purification of the hydrolysates was performed via ultrafiltration along with reversed-phase high performance liquid chromatography (RP-HPLC). Determination of the purified peptide amino acid sequence was confirmed for both peptides and reported as Ala-Phe-Asn-Arg-Ala-Lys-Ser-Lys-Ala-Leu-Asn-Glu-Asn (AFNRAKSKALNEN; Basil-1), and Tyr-Asp-Ser-Ser-Gly-Gly-Pro-Thr-Pro-Trp-Leu-Ser-Pro-Tyr (YDSSGGPTPWLSPY; Basil-2). The respective activities for calcium-binding were 38.62 ± 1.33%, and 42.19 ± 2.27%. Fluorescence spectroscopy, and fourier transform infrared spectroscopy were employed in order to assess the chelating mechanism between calcium and the peptides. It was found that the calcium ions took place through the activity of the amino nitrogen atoms and the oxygen atoms on the carboxyl group. Moreover, both of these peptides served to improve calcium transport and absorption in Caco-2 cell monolayers, depending on the concentration involved. It was revealed that the peptide-calcium complexes offered an increased calcium absorption percentage when compared to free calcium at similar concentrations. It might be concluded that the peptide within the peptide-calcium complex can promote calcium absorption through both active and passive transport pathways by increasing calcium concentration and promoting cell membrane interaction. Accordingly, DLBS protein can be considered a strong potential source of protein which can be used to produce calcium-binding peptides and might therefore play a role in the production of nutraceutical foods as a bioactive ingredient.

Microalgae as Potential Sources of Bioactive Compounds for Functional Foods and Pharmaceuticals

“…This means that chelating compounds were concentrated in the hexane fractions, suggesting that the compounds with calcium-chelating capacity are from the same polarity family, since non-polar solvents tend to extract non-polar compounds (such as fatty acids, carotenoids, and sterols, among others). It has been reported that compounds with amino, carboxyl, and phosphoric groups may be responsible for the calcium-binding activity of compounds due to simultaneously providing an atom with a non-bonding free electron pair (nitrogen, oxygen or phosphorus) and a suitable binding site [49]. Our findings suggest that both hexane fractions may be sources of calcium chelating compounds, and could play a significant role in neuroprotective mechanisms as evidence suggests that an intracellular calcium overload is linked to the neurobiology of neurodegenerative diseases (NDs) like Alzheimer's [50,51].…”

The Formation, Structural Characteristics, Absorption Pathways and Bioavailability of Calcium–Peptide Chelates

“…Wang et al extracted a peptide with a CaBC of 191.5 µg/mg from cucumber seeds, which the authors asserted could be used as a new calcium supplement [ 17 ]. Furthermore, two CaBP with the sequences of AFNRAKSKALNEN and YDSSGGPTPWLSPY were isolated from lemon basil seeds, with CaBCs of 278.14 µg/mg and 151.88 µg/mg, respectively [ 18 ]. Chlorella is a single-celled green algae that comprises more than 50% protein.…”

“…These two conclusions demonstrated the combination of hydroxyl oxygen and carboxyl oxygen with the calcium ions [ 20 ]. In another assay, the infrared spectra of lemon basil seed peptides revealed that the amide I band (which referred to the carbonyl group absorption peak) shifted from 1624.34 cm −1 to 1626.85 cm −1 after binding to calcium ions, and a new peak appeared in the range of 1500–1400 cm −1 after chelation, suggesting that both oxygen atoms of the carboxyl group were involved in the binding [ 18 ]. Liu et al found that the infrared spectra of wheat peptides bound to calcium ions was shifted in the region of 1658 cm −1 to 1650 cm −1 by C=O stretching vibrations, thus proving that the carbonyl group had combined with the calcium ions.…”

“…Cell viability reached its highest value at 50 μg/mL and increased by 35% compared with the control group, thereby indicating that the EWP-Ca was not cytotoxic [ 78 ]. In addition, Lin et al proved that the viability of Caco-2 cells treated with snapper fish scale protein peptide and CaCl 2 was greater than 85% [ 42 ], and a chelate formed by basil seed peptides and calcium ions showed similar properties [ 18 ]. In combination, none of the different sources of peptide chelates with calcium ions have been reported to exhibit cytotoxicity.…”

Osteogenic activities of four calcium‐chelating microalgae peptides

“…16 Peptides from defatted lemon basil seeds can chelate calcium to form 'peptide-calcium complexes', promoting calcium absorption and transport in Caco-2 cells. 17 Microalgae are ideal materials for producing bioactive peptides because of their low cost, high production and high protein content. They contain abundant amino acids comparable to some recognized high-protein food like soybeans and eggs.…”