Preparation, Characterization, and Antioxidant Properties of Selenium-Enriched Tea Peptides

Abstract

The research on the activity of selenium (Se)-enriched agricultural products is receiving increasing attention since Se was recognized for its antioxidant activities and for its enhancement of immunity in trace elements. In this study, antioxidant Se-containing peptides, namely, Se-TAPepI-1 and Se-TAPepI-2, were optimally separated and prepared from Se-enriched tea protein hydrolysates by ultrafiltration and Sephadex G-25 purification, and subsequently, their physicochemical properties, oligopeptide sequence, and potential antioxidant mechanism were analyzed. Through the optimization of enzymatic hydrolysis conditions, the Se-enriched tea protein hydrolyzed by papain exhibited a better free radical scavenging activity. After separation and purification of hydrolysates, the two peptide fractions obtained showed significant differences in selenium content, amino acid composition, apparent morphology, peptide sequence, and free radical scavenging activity. Therein, two peptides from Se-TAPepI-1 included LPMFG (563.27 Da) and YPQSFIR (909.47 Da), and three peptides from Se-TAPepI-2 included GVNVPYK (775.42 Da), KGGPGG (552.24 Da), and GDEPPIVK (853.45 Da). Se-TAPepI-1 and Se-TAPepI-2 could ameliorate the cell peroxidation damage and inflammation by regulating NRF2/ARE pathway expression. Comparably, Se-TAPepI-1 showed a better regulatory effect than Se-TAPepI-2 due to their higher Se content, typical amino acid composition and sequence, higher surface roughness, and a looser arrangement in their apparent morphology. These results expanded the functional activities of tea peptide and provided the theoretical basis for the development of Se-containing peptides from Se-enriched tea as a potential natural source of antioxidant dietary supplements.

Keywords: antioxidant activity; identification; selenium; tea peptide; tea protein.

Figure 1

Effects of different hydrolases on the free radical scavenging activities of Se-enriched alkali-soluble tea protein hydrolysate, including DPPH• (a), ABTS• (b), •OH (c), and O₂⁻ (d). Effects of pH (e), hydrolysis temperature (f), enzyme concentration (g), and hydrolysis time (h) on the •OH scavenging. Response surface plots for (i) Y = f (B, A), (j) Y = f (C, A), (k) Y = f (D, A), (l) Y = f (C, B), (m) Y = f (D, B), and (n) Y = f (D, C). Vc is vitamin C and mean values with different letters (a, b …) were significantly different in experiment (p < 0.05).

Figure 2

Effects of ultrafiltration components Se-TAPepI (<1 kda), Se-TAPepII (1–10 kda), Se-TAPep3III (10–100 kda) on the scavenging activities of DPPH• (a) and •OH (b). The UV scanning analysis of Se-TAPepI (c). The gel elution curve of Se-TAPepI (d). Effects of Se-TAPepI-1 and Se-TAPepI-2 on the scavenging activities of DPPH• (e) and •OH (f).

Figure 3

Fourier transform infrared spectra analysis of Se-TAPepI-1 and Se-TAPepI-2 (a). Scanning electron microscope of Se-TAPepI-1 and Se-TAPepI-2; the scale is marked in the figure (scale bar = 1 μm) (b).

Figure 4

Mass spectrum analysis of the antioxidant peptide derived from Se-TAPepI-1. Mass spectrum of GVNVPYK (a); mass spectrum of KGGPHG (b), the red and blue lines represent the two fragmented forms of secondary mass spectrometry of the target peptide chain.

Figure 5

Mass spectrum analysis of the antioxidant peptide derived from Se-TAPepI-2. Mass spectrum of LPMFG (a); mass spectrum of YPQSFIR (b); mass spectrum of GDEPPIVK (c), the red and blue lines represent the two fragmented forms of secondary mass spectrometry of the target peptide chain.

Figure 6

Protection of Se-TAPepIs in H₂O₂-induced oxidatively injured LO2 cells. The effect of Se-TAPepIs on cell proliferation in H₂O₂-induced oxidatively injured LO2 cells (a). The effect of Se-TAPepI-1 and Se-TAPepI-2 on ROS production in H₂O₂-induced oxidatively injured LO2 cells (b). Images of ROS production observed by fluorescence microscopy (200×) (c). The enzyme activity of SOD (d), GSH-Px (e), and CAT (f). ns indicates p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were compared with the MC. MC (model control) indicates LO2 cells with oxidative damage, and NC (normal control) refers to untreated LO2 cells.

Figure 7

The effects of Se-TAPepIs on mRNA expression and protein levels in H₂O₂-induced oxidatively injured LO2 cells. The mRNA expression of NRF2 (a), HO-1 (b), IL-1β (c), TNF-α (d) in each treated group of LO2 cells. The protein levels of NRF2, HO-1, NQO-1 and GCLC (e) and its grayscale images (f) in each treated group of LO2 cells. ns indicates p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were compared with the MC. MC (model control) indicates LO2 cells with oxidative damage, and NC (normal control) refers to untreated LO2 cells and mean values with different letters (a, b …) were significantly different in experiment (p < 0.05).