Preparation, Characterization, and Mechanism of Antifreeze Peptides from Defatted Antarctic Krill (Euphausia superba) on Lactobacillus rhamnosus

Abstract

Defatted Antarctic krill powder is the main by-product in the manufacturing of krill oil. Exploring a high value-added approach for utilizing this protein-rich material has received much attention in research and industry. Given this, the preparation and primary characterization of antifreeze peptides from defatted Antarctic krill (AKAPs) were carried out in this study. The cryoprotective effect of AKAPs on Lactobacillus rhamnosus ATCC7469 was also investigated. The results showed that Protamex was the optimum protease for AKAP preparation from defatted Antarctic krill. AKAPs were found to be rich in short peptides, with the MW ranging from 600 to 2000 Da (69.2%). An amino acid composition analysis showed that AKAPs were rich in glutamic acid (18.71%), aspartic acid (12.19%), leucine (7.87%), and lysine (7.61%). After freezing, the relative survival rate of Lactobacillus rhamnosus in the 1.0 mg/mL AKAP-treated group (96.83%) was significantly higher than in the saline group (24.12%) (p < 0.05). AKAPs also retarded the loss of acidifying activity of L. rhamnosus after freezing. AKAPs showed even better cryoprotective activity than three commercial cryoprotectants (sucrose, skim milk, and glycerol). In addition, AKAPs significantly alleviated the decrease in β-galactosidase and lactic dehydrogenase activities of L. rhamnosus (p < 0.05). Furthermore, AKAPs effectively protected the integrity of L. rhamnosus cell membranes from freezing damage and alleviated the leakage of intracellular substances. These findings demonstrate that AKAPs can be a potential cryoprotectant for preserving L. rhamnosus, providing a new way to use defatted Antarctic krill.

Keywords: Antarctic krill; Lactobacillus rhamnosus; antifreeze peptide; by-products; cryoprotectant; freezing.

Figure 1

Cryoprotective activities of enzymatic hydrolysates from defatted Antarctic krill at a 1.0 mg/mL concentration treated with different proteases. Bars with different lowercase letters (a, b, and c) indicate significant differences (p < 0.05).

Figure 2

(A) HPLC elution of molecular weight and (B) molecular weight distribution of antifreeze peptides from defatted Antarctic krill (AKAPs).

Figure 3

Effects of the negative control (saline), positive controls (1 mg/mL sucrose, 1 mg/mL skim milk, and 20% glycerol), 0.5 mg/mL antifreeze peptides from defatted Antarctic krill (AKAP) (L-AKAP), and 1 mg/mL AKAP (H-AKAP) on the (A) growth and (B) pH values of L. rhamnosus after freezing. The unrefrigeration group in (B) did not experience the freezing process. Bars with different lowercase letters (a, b, c, and d) indicate significant differences (p < 0.05).

Figure 4

Effects of the negative control (saline), positive controls (1 mg/mL sucrose, 1 mg/mL skim milk, and 20% glycerol), 0.5 mg/mL antifreeze peptides from defatted Antarctic krill (AKAPs) (L-AKAP), and 1 mg/mL AKAP (H-AKAP) on the (A) β-galactosidase (β-GAL) activity and (B) lactic dehydrogenase (LDH) activity of L. rhamnosus after freezing. Bars with different lowercase letters (a, b, and c) indicate significant differences (p < 0.05).

Figure 5

Effects of the negative control (saline), positive controls (1 mg/mL sucrose, 1 mg/mL skim milk, and 20% glycerol), 0.5 mg/mL antifreeze peptides from defatted Antarctic krill (AKAPs) (L-AKAP), and 1 mg/mL AKAP (H-AKAP) on concentrations of extracellular protein of L. rhamnosus after freezing. Bars with different lowercase letters (a, b, c, and d) indicate significant differences (p < 0.05).

Figure 6

Representative scanning electron micrographs of (A) unrefrigerated L. rhamnosus, (B) saline-treated L. rhamnosus after freezing, and (C) L. rhamnosus treated with 1 mg/mL antifreeze peptides from defatted Antarctic krill (AKAPs) (H-AKAP) after freezing. (1) represents 20,000-fold magnifications and (2) represents 70,000-fold magnifications.