Antifreeze Peptides Preparation from Tilapia Skin and Evaluation of Its Cryoprotective Effect on Lacticaseibacillus rhamnosus

Abstract

Antifreeze peptides can protect cell membranes and maintain the cell viability of probiotics under cold stress. Given this, antifreeze peptides were prepared from tilapia processing byproducts of tilapia skin by enzymolysis using the response surface methodology (RSM) method. The cryoprotective effects on Lacticaseibacillus rhamnosus ATCC7469 were investigated. Trypsin was selected as the protease for tilapia skin hydrolysis. The optimal hydrolysis conditions consisted of the amount of enzyme (2200 U/g), solid-liquid ratio (1:10, w/v), reaction temperature (49 °C), and reaction time (6.8 h), and the relative survival rate of L. rhamnosus reached 98.32%. Molecular weight (M_w) distribution and peptide sequences of the antifreeze peptides prepared from tilapia skin (APT) under the optimal conditions were analyzed. APT significantly reduced the leakage of extracellular proteins and protected β-galactosidase and lactate dehydrogenase activities of L. rhamnosus. Compared with the saline group, scanning electron microscopy (SEM) observation showed that cells had a more normal, smooth, and entire surface under the protection of APT. These findings indicate that APT can be a new cryoprotectant in preserving probiotics.

Keywords: Lacticaseibacillus rhamnosus ATCC7469; antifreeze peptide; cryoprotective mechanism; tilapia skin.

Figure 1

The antifreeze activities of different enzymatic hydrolysis samples (1 mg/mL), positive controls (1 mg/mL sucrose, 1 mg/mL skim milk, and 20% (v/v) glycerol), and negative control (sterilized saline) on L. rhamnosus frozen at −20 °C for 24 h. The bars with different letters indicate significant differences (p < 0.05).

Figure 2

The interaction of independent variables on the relative survival rate of L. rhamnosus frozen at −20 °C for 24 h. The interaction between two independent variables were as follows: (A), the solid–liquid ratio and amount of enzyme; (B), the reaction temperature and amount of enzyme; (C), the reaction time and amount of enzyme; (D), the reaction temperature and solid–liquid ratio; (E), the reaction time and solid–liquid ratio; (F), the reaction temperature and reaction time.

Figure 3

The molecular weight distribution of APT.

Figure 4

Effects of saline, positive cryoprotectants, and APT on β-galactosidase (β-GAL) and lactic dehydrogenase (LDH) activities of L. rhamnosus frozen at −20 °C for 24 h. The bars in the same color with different capital letters or lowercase letters indicate significant differences (p < 0.05).

Figure 5

Effects of saline, positive cryoprotectants, and APT on the proteins leakage of L. rhamnosus after frozen at −20 °C for 24 h. The bars with different lowercase letters indicate significant differences (p < 0.05).

Figure 6

The scanning electron micrographs of L. rhamnosus frozen at −20 °C for 24 h with saline (left) and APT (right).