Oyster-Derived Zinc-Binding Peptide Modified by Plastein Reaction via Zinc Chelation Promotes the Intestinal Absorption of Zinc

Abstract

Zinc-binding peptides from oyster (Crassostrea gigas) have potential effects on zinc supplementation. The aim of this study was to prepare efficient zinc-binding peptides from oyster-modified hydrolysates by adding exogenous glutamate according to the plastein reaction and to further explore the zinc absorption mechanism of the peptide-zinc complex (MZ). The optimum conditions for the plastein reaction were as follows: pH 5.0, 40 °C, substrate concentration of 40%, pepsin dosage of 500 U/g, reaction time of 3 h and l-[1-¹³C]glutamate concentration of 10 mg/mL. The results of ¹³C isotope labelling suggested that the addition of l-[1-¹³C]glutamate contributed to the increase in the zinc-binding capacity of the peptide. The hydrophobic interaction was the main mechanism of action of the plastein reaction. Ultraviolet spectra and scanning electronic microscopy (SEM) revealed that the zinc-binding peptide could bind with zinc and form MZ. Furthermore, MZ could significantly enhance zinc bioavailability in the presence of phytic acid, compared to the commonly used ZnSO₄. Additionally, MZ significantly promoted the intestinal absorption of zinc mainly through two pathways, the zinc ion channel and the small peptide transport pathway. Our work attempted to increase the understanding of the zinc absorption mechanism of MZ and to support the potential application of MZ as a supplementary medicine.

Keywords: caco-2 cells; intestinal absorption; oyster zinc-binding peptide; peptide-zinc complex; zinc bioavailability.

Figure 1

Effects of hydrolysis pH (A), temperature (B), substrate concentration (C), pepsin dosage (D), reaction time (E), and glutamate concentration (F) on free amino acids reduction of the plastein products. Unless otherwise noted, temperate was 40 °C, pH was 5.0, substrate concentration was 40%, pepsin dosage was 500 U/g, reaction time was 3 h, and l-[1-¹³C]glutamate concentration was 10 mg/mL. Each point is shown as the means ± standard deviation (SD) (n = 3). Different letters indicate significant differences (p < 0.05).

Figure 2

Change in hydrophobicity (A) and zinc-binding capacity (B) during the plastein reaction. Each point is shown as the means ± SD (n = 3). Different letters indicate significant differences (p < 0.05).

Figure 3

(A) Solubility of plastein products in different denaturants; (B) effect of urea on the stability of plastein products. Abbreviations: DW, deionized water; NaCl, sodium chloride; TCA, trichloroacetic acid; HAc, acetic acid; SDS, sodium dodecyl sulfate. Each point is shown as the means ± SD (n = 3). Asterisk (*) and different letters indicate significant differences (p < 0.05).

Figure 4

The change of molecular weight distribution during plastein reaction. Each point is shown as the means ± SD (n = 3). Asterisk (*) indicate significant differences (p < 0.05).

Figure 5

Sephadex G-15 chromatograph of phenylalanine standard (A) and different components of plastein products (B), a–d mean the fractions that obtained at 220 nm.

Figure 6

Scanning electron microscopy (SEM) photograph of peptide (A,C) and peptide-zinc complex (MZ) (B,D), UV-vis absorption analysis (E).

Figure 7

(A) Cell viability of Caco-2 cells after being incubated with the MZ at different concentrations. (B) Zinc uptake by Caco-2 cell monolayers in the presence of 2:1 molar ratio of zinc/phytic acid. Cellular uptake of exogenous zinc was measured as the quenching of Zinquin ethyl ester fluorescence. (C) The free zinc content after phytic acid treatment. M: peptide (EVPPEEH); MZ: peptide-zinc complex; AP: phytic acid. Different lowercase letters and asterisk (*) indicates significant difference between groups (p < 0.05).

Figure 8

Effects of MZ on the relative level of hZIP4, ZnT1, and PepT1 mRNA in Caco-2 cells. MZ = peptide-zinc complex. Asterisk (*) indicates significant difference between groups (p < 0.05).

Figure 9

Possible mechanisms of MZ promotes zinc absorption of Caco-2 cells.