Bioactive Peptides from Skipjack Tuna Cardiac Arterial Bulbs: Preparation, Identification, Antioxidant Activity, and Stability against Thermal, pH, and Simulated Gastrointestinal Digestion Treatments

Abstract

Cardiac arterial bulbs of Skipjack tuna (Katsuwonus pelamis) are rich in elastin, and its hydrolysates are high quality raw materials for daily cosmetics. In order to effectively utilizing Skipjack tuna processing byproducts-cardiac arterial bulbs and to prepare peptides with high antioxidant activity, pepsin was selected from six proteases for hydrolyzing proteins, and the best hydrolysis conditions of pepsin were optimized. Using ultrafiltration and chromatographic methods, eleven antioxidant peptides were purified from protein hydrolysate of tuna cardiac arterial bulbs. Four tripeptides (QGD, PKK, GPQ and GLN) were identified as well as seven pentapeptides (GEQSN, GEEGD, YEGGD, GEGER, GEGQR, GPGLM and GDRGD). Three out of them, namely the tripeptide PKK and the pentapeptides YEGGD and GPGLM exhibited the highest radical scavenging activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and superoxide anion assays. They also showed to protect plasmid DNA and HepG2 cells against H₂O₂-induced oxidative stress. Furthermore, they exhibited high stability under temperature ranged from 20-100 °C, pH values ranged from 3-11, and they simulated gastrointestinal digestion for 240 min. These results suggest that the prepared eleven antioxidant peptides from cardiac arterial bulbs, especially the three peptides PKK, YEGGD, and GPGLM, could serve as promising candidates in health-promoting products due to their high antioxidant activity and their stability.

Keywords: Skipjack tuna (Katsuwonus pelamis); antioxidant activity; cardiac arterial bulbs; peptide; stability.

Figure 1

Influences of protease species on DPPH· (A) and HO· (B) scavenging activity of protein hydrolysates from Skipjack tuna cardiac arterial bulbs at 2.0 mg/mL. ^a–f Values with same letters indicate no significant difference (p > 0.05).

Figure 2

Effects of enzyme concentration (A), material/liquid ratio (B), and hydrolysis time (C) of pepsin on DPPH· scavenging activity of protein hydrolysates from tuna cardiac arterial bulbs at 2.0 mg/mL. ^a–e Values with same letters indicate no significant difference (p > 0.05).

Figure 3

Response surface graph for DPPH· scavenging activity (%) as a function of (A) hydrolysis time (X₁) and material/liquid ratio (X₂), (B) hydrolysis time (X₁) and enzyme concentration (X₃), (C) material/liquid ratio (X₂) and enzyme concentration (X₃) during the hydrolysis of Skipjack tuna cardiac arterial bulbs with pepsin.

Figure 4

Radical scavenging activity of pepsin hydrolysate of Skipjack tuna cardiac arterial bulbs (TCAH) and its four fractions (TCAH-I to TCAH-IV). ^a–c Values and ^A–C values with same letters indicate no significant difference (p > 0.05).

Figure 5

Chromatogram profile of TCAH-I isolated by DEAE-52 cellulose (A) and the radical scavenging activity of prepared subfractions (IEC-I to IEC-IV) from TCAH-I at 2 mg/mL (B). ^a–d Values and ^A–D values with same letters indicate no significant difference (p > 0.05).

Figure 6

Chromatogram profile of IEC-II isolated by Sephadex G-25 (A) and the radical scavenging activity of prepared subfractions (GPC-I and GPC-II) from IEC-II at 2 mg/mL (B). ^a–c Values and ^A–C values with same letters indicate no significant difference (p > 0.05).

Figure 7

Elution profiles of GPC-II by RP-HPLC at 220 nm (A) and 254 nm (B).

Figure 8

Mass spectrogram of 11 APs (TCP1 to TCP11) from hydrolysate of Skipjack tuna cardiac arterial bulbs (TCAH). (A) TCP1; (B) TCP2; (C) TCP3; (D) TCP4; (E) TCP5; (F) TCP6; (G) TCP7; (H) TCP8; (I) TCP9; (J) TCP10; (K) TCP11.

Figure 9

Protective effects of TCP3, TCP6, and TCP9 on H₂O₂-damaged plasmid DNA (pBR322DNA). Lane 1: the native pBR322DNA; Lane 2, DNA + FeSO₄ + H₂O₂ + TCP9 (200 μM); Lane 3, DNA + FeSO₄ + H₂O₂ + TCP6 (200 μM); Lane 4, DNA + FeSO₄ + H₂O₂ + TCP3 (200 μM); Lane 5, DNA + FeSO₄ + H₂O₂ + TCP9 (100 μM); Lane 6, DNA + FeSO₄ + H₂O₂ + TCP6 (100 μM); Lane 7, DNA + FeSO₄ + H₂O₂ + TCP3 (100 μM); Lane 8, pBR322DNA + FeSO₄ + H₂O₂.

Figure 10

Effects of TCP3, TCP6, and TCP9 on the viability of H₂O₂-damaged HepG2 cells. N-Acetyl-L-Cysteine (NAC) was served as the positive control. ^### p < 0.001 vs. blank control group; *** p < 0.001 vs. model group.

Figure 11

DPPH· scavenging activity of TCP1–TCP11 subjected to different thermal treatments for 60 min. ^a–d Values with same letters indicate no significant difference of same peptide (p > 0.05).

Figure 12

DPPH· scavenging activity of TCP1–TCP11 subjected to different pH treatments for 60 min (A), 120 min (B), and 180 min (C), respectively. ^a–c values with same letters indicate no significant difference of same peptide (p > 0.05).

Figure 13

DPPH· scavenging activity of TCP1–TCP11 subjected to simulated GI digestion treatments from 0 to 240 min. (A): TCP1, TCP5, TCP7, and TCP10; (B): TCP2, TCP4, TCP8, and TCP11; (C): TCP3, TCP6, and TCP9. ^a–e Values with same letters indicate no significant difference of same peptide (p > 0.05).

Figure 14

Flow diagram of isolating APs from protein hydrolysate (TCAH) of tuna cardiac arterial bulbs prepared by pepsin.