Potential of Marine Bacterial Metalloprotease A69 in the Preparation of Antarctic Krill Peptides with Multi-Bioactivities

Abstract

Antarctic krill (Euphausia superba) is a nutrient-rich marine resource. Although several terrestrial proteases have been used to prepare Antarctic krill peptides (AKPs), there has been no report on the preparation of AKPs using a marine protease. Here, marine bacterial protease A69 was used to prepare AKPs with multi-bioactivities. Through optimizing hydrolysis parameters, we established a process for AKPs preparation by hydrolyzing Antarctic krill powder with A69. In the prepared AKPs, peptides less than 3000 Da and 1000 Da accounted for 99.23% and 88.37%, respectively. The scavenging ratios of the AKPs to ABTS⁺, DPPH· and ·OH reached 93.23 ± 0.09%, 99.90 ± 0.15%, and 93.90 ± 0.47%, respectively. The AKPs also had high angiotensin-converting enzyme (ACE)-inhibitory activity, with an IC₅₀ of 0.22 ± 0.04 mg/mL. At 40 mg/mL, the AKPs inhibited α-glucosidase and dipeptidyl peptidase IV (DPP-IV) activities by 7.18% and 13.62%, respectively, and displayed antibacterial activity to Escherichia coli. Moreover, 14 antioxidant peptides, 24 ACE-inhibitory peptides, 2 α-glucosidase-inhibitory peptides, and 10 DPP-Ⅳ-inhibitory peptides were identified from the AKPs. These results demonstrate that the prepared AKPs contain diverse bioactive peptides and have multi-bioactivities. This study indicates that marine bacterial protease A69 has promising application potential in preparing AKPs with multi-bioactivities.

Keywords: Antarctic krill peptides; angiotensin-converting enzyme (ACE)-inhibitory activity; antibacterial activity; antioxidant activity; dipeptidyl peptidase IV (DPP-IV)-inhibitory activity; marine bacterial protease A69; α-glucosidase-inhibitory activity.

Figure 1

Optimization of the parameters for protease A69 to hydrolyze Antarctic krill powder. (A) The effect of the E/S ratio on the hydrolysate yield. Antarctic krill powder was hydrolyzed at 60 °C and pH 7.0 for 6 h by A69 under different E/S ratios. (B) The effect of hydrolysis time on the hydrolysate yield. Antarctic krill powder was hydrolyzed at the E/S ratio of 5000 U/g, 60 °C, and pH 7.0 for different time durations. The graphs show data from triplicate experiments (mean ± SD).

Figure 2

A flow chart of AKPs preparation with protease A69.

Figure 3

Antarctic krill powder (A) and the AKPs powder (B). The AKPs powder was prepared using the process shown in Figure 2.

Figure 4

Solubility of the prepared AKPs powder. (A) The AKPs solution of 10% (w/v) concentration. (B) The AKPs solution of 20% (w/v) concentration. (C) The AKPs solution of 30% (w/v) concentration.

Figure 5

Molecular weight distribution of the prepared AKPs analyzed by HPLC gel filtration.

Figure 6

Antioxidant activity of the prepared AKPs. (A) ABTS⁺ scavenging capacity of the AKPs. (B) •OH scavenging capacity of the AKPs. (C) DPPH• scavenging capacity of the AKPs. (D) O₂⁻• scavenging capacity of the AKPs. The graphs show data from triplicate experiments (mean ± SD).

Figure 7

The ACE-inhibitory activity of the AKPs. The graph shows data from triplicate experiments (mean ± SD).

Figure 8

Antibacterial effects of the AKPs on E. coli (A) and S. aureus (B). Plates in the figure are representatives from triplicate experiments.