Snow flea antifreeze peptide for cryopreservation of lactic acid bacteria

Expression, purification, and identification of rsfAFP

DNA sequence and the corresponding translated peptide sequence is shown in Supplementary Fig. 1. The recombinant plasmid pHT43-SF-P was introduced into B. subtilis strains by electroporation. Recombinant B. subtilis clones were identified by PCR and the results are presented in Fig. 1A. The results showed that all single clones were positive clones with the correctly sized 543 bp target band (Fig. 1A). These results indicated that the target genes successfully recombined into pHT43, as anticipated.

A PCR detects positive clones. M: DNA marker DL10000; lanes 1–7: recombinant plasmid; +: positive control. B Expression result. Coomassie blue staining. MW: molecular weight marker. ϕ: Transformed cells not induced. C Western blot detects expression results. MW: molecular weight marker. ϕ: transformed cells not induced. +: positive control. D Target protein purification profile. IN: input; FT: flow through; MW: molecular weight marker; W1– W3: wash with TBS buffer with 0, 30, and 50 mM imidazole, respectively; E1–E5: eluted with TBS buffer with 100, 150, 200, 250, and 400 mM imidazole, respectively. E Sequence cover situation of rsfAFP by Nano LC-MS/MS spectrometry. The red section indicates the peptides matching the peptides of the snow flea antifreeze protein. The gray section indicates the missing fragments in the Nano LC-MS/MS test. The green section of peptide is GS linker. The blue section of peptide is 6*his-tag.

After harvesting the culture medium, the proteins were analyzed by SDS-PAGE and western blot (Fig. 1B, C). The results showed that the target protein was expressed by the WB800N strain. The optimal conditions of expression were: 30 °C, 4 h, and 1 mM isopropyl β–d-1-thiogalactopyranoside (IPTG). The target protein was further isolated and purified by affinity Ni-Charged resin chromatography with a final purity of over 95%. SDS-PAGE displayed a clear single target protein band (Fig. 1D). The band of the target protein in the SDS-PAGE was excised and then submitted to in-gel digestion with trypsin. The amino acid sequence of the target protein was carried out using nano liquid chromatography-tandem mass spectrometry (Nano LC-MS/MS; Fig. 1E). The sequence coverage of the target protein was analyzed by MASCOT software analysis, and the sequence coverage was 90.86%. Therefore, through PCR, western blot, and Nano LC-MS/MS, it was demonstrated that the target protein (rsfAFP) was successfully constructed in the B. subtilis WB800N expression system.

Antifreeze activities of rsfAFP

AFPs are unique molecules that can bind at the growth interface of ice and are active on the ice-water interface. This property of AFPs mainly depends on their THA and IRI ability²⁶. TH is due to the adhesion of AFPs on the ice surface, increasing the growth curvature of the ice crystals. This results in an increase in the vapor pressure of the ice-water interface layer, requiring greater energy for the ice crystal growth. THA is the gap between the freezing and melting points of a solution, and it is an important parameter to evaluate the activity of AFPs. Compared with the standard proteins (bovine serum albumin (BSA)) in the control groups, rsfAFP showed a significant TH exothermic peak (Fig. 2A, B). Through integral calculation, it was found that under the same holding temperature, the THA of rsfAFP increases significantly compared to BSA (Fig. 2C). Although the THA of rsfAFP is not excellent, the ice crystal content was significantly reduced after the addition of rsfAFP (Fig. 2D).

The DSC thermogram of the freezing and melting processes at different holding temperatures for A BSA and B rsfAFP. C The thermal hysteresis activity of BSA and rsfAFP at different holding temperatures. D The ice content of BSA and rsfAFP at different thermal hysteresis. Data points represent mean values calculated from two separate experiments.

The frost-related damage of microorganisms during storage under freezing conditions is mainly caused by the recrystallization and thawing process^2,27. Therefore, compared with THA, the IRI ability of AFPs is more important for frozen products during storage. The preferential adsorption of AFPs on the growth interface of ice to control the ice growth is widely accepted as conferring them their IRI ability and functional role in protecting organisms from freezing damage^14,27,28,29. The IRI of rsfAFP was conducted via a polarized light microscope with a cold stage, and the results are shown in Fig. 3. The ice crystals in the negative groups grew significantly, circular in shape after five freeze-thaw cycles (Fig. 3A). It is worth noting that the ice crystal size of the samples with rsfAFP was significantly smaller than that of the negative group (Fig. 3B). These results indicated that there may be a specific interaction between rsfAFP and ice, inhibiting the recrystallization of ice crystals.

A, B The effects of rsfAFP on ice recrystallization in PBS (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0) after different thermal cycles between −14 and −12 °C. A-1, A-3, and A-5 show the ice crystal growth without rsfAFP before, after 3 cycles and 5 cycles, respectively. B-1, B-3, and B-5 show the ice crystal growth with rsfAFP (0.1 mg/mL) before, after 3 cycles and 5 cycles, respectively. The morphology and growth rate of a single ice crystal without (C) or with (D) addition of 0.1 mg/mL rsfAFP in ultrapure water.

To demonstrate that rsfAFP can bind to the growth interface of ice crystals, the growth morphology of a single ice crystal was investigated using a nanoliter osmometer. As illustrated in Fig. 3C, in ultrapure water, ice is shaped like a flat disk and grows so rapidly that it takes only 20 s for the ice to fill the entire field of observation. Interestingly, the growth rate of ice crystals was significantly inhibited in rsfAFP aqueous dispersion, and the morphology of ice was significantly isomerized (Fig. 3D). The hexagonal shape of the ice is attributed to the fact that rsfAFP binds to the prism plane of ice^30,31,32. The identification of antifreeze activity was demonstrated by the ability of rsfAFP to significantly regulate ice crystals.

rsfAFP cytoprotection affects the physiological functions of S. thermophilus under freezing stress

To evaluate the feasibility of rsfAFP as a substitute for current commercial cryoprotectants, a series of experiments were undertaken on the cryoprotective of rsfAFP on S. thermophilus. Different cryoprotectants were added to S. thermophilus and then exposed to freeze–thaw cycling models. The physiological functions of S. thermophilus with different cryoprotective formulations after freezing stress are shown in Fig. 4.

A The survival rate of S. thermophilus after freezing at −20 °C for 24 h and 2 freezing–thawing cycles with various cryoprotectants. B S. thermophilus metabolic activity; C acid production of S. thermophilus with different cryoprotectants; D S. thermophilus growth with various cryoprotectants at different freezing times. The concentration of rsfAFP was 0.1 mg/mL. 20 mM PBS was used as a negative control; 15% glycerol, 1.0 mg/mL sucrose, and 1.0 mg/mL skim milk were used as the positive controls.

The survival rate of cryopreserved S. thermophilus thawed at 37 °C with different cryoprotectants is shown in Fig. 4A. The survival rate of S. thermophilus with 0.1 mg/mL rsfAFP was greatly increased from 8.86% to 93.21%, which was much higher than that observed when 1.0 mg/mL sucrose (60.71%), 1.0 mg/mL skim milk (65.39%), and 15% glycerol (79.80%) were used as cryoprotectants. During the freezing process, the extracellular water was first freezing into ice. The pressure difference between the inside and outside caused dehydration of the cell, increasing the intracellular electrolyte concentration. This elevated intracellular solute led to the denaturation or inactivation of sensitive enzymes as well as certain proteins, affecting the metabolic activity of cells. The metabolic vitality of S. thermophilus with different cryoprotectants is shown in Fig. 4B. The metabolic activity of the phosphate buffer solution (PBS), sucrose, skim milk, glycerol, and rsfAFP groups were 28.34, 50.04, 62.65, 71.06, and 82.54%, respectively. These results showed that the metabolic activity of S. thermophilus in the rsfAFP group was greatly improved when compared with the commercial cryoprotectant groups.

The acid production capacity is related to the metabolic activity of S. thermophilus cells, so the cryoprotective effect of different cryoprotectants on bacterial cells can be reflected by measuring the acid production. The acid production and the growing stability of S. thermophilus with different cryoprotectants after freezing stress are shown in Fig. 4C, D, respectively. Consistent with the trends for the survival rate, the acid production of rsfAFP group was significantly higher than that in commercial cryoprotectant groups. In addition, rsfAFP added to S. thermophilus significantly improved the freezing stability. After 14 days under freezing conditions, the survival rate in the PBS group decreased by 97.88%. The survival rate in the glycerol group, the most commonly used commercial cryoprotectant (glycerol group), still decreased by 61.57%. However, the survival rate of the rsfAFP group decreased only by 38.50% under the same storage conditions.

Micromorphological characteristics of S. thermophilus cells

SEM observations revealed that the S. thermophilus cells without cryoprotectants were significantly damaged during frozen storage, and most of the cells had no intact cellular structure after freezing stress treatment. In addition, due to the rupture of the cell structure, the contents of S. thermophilus cells leaked out and deposited on the surface of the cells, making the cells adhere to each other without clear boundaries (Fig. 5). The cellular morphology in the two commercial cryoprotectants groups (sucrose and glycerol) was significantly improved when compared to the negative control group, but still showed varying degrees of damage. As anticipated, under the protection of rsfAFP, the cells were full, round and still presented a complete cell structure. Mechanical stress damage to S. thermophilus cells caused by the formation of big ice crystals during freezing storage is the main culprit for cell rupture. Previous studies on cell viability found that cell survival rate decreased after cryopreservation, likely caused by mechanical damage, and resulted in the inability of cells to fully recover. Therefore, rsfAFP can protect cells from the mechanical stress damage caused by big ice crystals during freezing storage by inhibiting the growth of ice crystals.

The concentration of rsfAFP was 0.1 mg/mL. 20 mM PBS was used as s negative control; 15% glycerol, 1.0 mg/mL sucrose were used as the positive controls. Green arrow: damaged cells; red arrow: ruptured cells.

Fourier transform infrared (FTIR) spectroscopic analysis

There is normally a lot of water on the surface of the cell membrane. The hydration layer formed between the polar head of the phospholipid and the water molecules around the cell membrane helps maintain the fluidity of the cell membrane. During the freezing or freeze-drying process, the hydration layer is destroyed, causing the phospholipid bilayer to change from a disordered liquid crystal phase to a rigid and ordered gel phase³³. This change can cause serious damage to the cell membrane. Since lecithin is one of the important components that make up the specific structure of cell membranes, the interaction between rsfAFP and cell membrane can be simulated by measuring the interaction between rsfAFP and lecithin, allowing to explore the cryoprotective mechanism of rsfAFP on the cell membrane.

The FTIR spectra of egg yolk lecithin head groups have distinct bands in the six-spectrum region (Fig. 6). These bands were 2925.22 cm⁻¹, 1740.28 cm⁻¹, 1465.87 cm⁻¹, 1241.59 cm⁻¹, 1089.98 cm⁻¹, and 969.95 cm⁻¹, corresponding to the egg yolk lecithin -CH₂ stretching vibration, symmetric C=O stretching vibration, -CH₃ antisymmetric deformation vibration, symmetric P=O stretching vibration, asymmetric P-O-C stretching vibration, and C-C-N⁺ stretching vibration, respectively^34,35. Through the FTIR spectra analysis, it was found that the presence of rsfAFP changed the characteristic spectrum of lecithin head groups (Fig. 6). The wavenumbers of 1241.80 cm⁻¹ and 969.95 cm⁻¹ show redshift, indicating that the P=O group and C-C-N⁺ group of lecithin are unstable, and transfer to high wavenumber after combining with rsfAFP. In other words, the energy required for vibration after binding becomes larger and the group becomes more stable.

A Characteristic FTIR spectra of egg yolk lecithin head; B FTIR spectra of egg yolk lecithin, rsfAFP, and their mixture.

The hydrogen bonds between the polar head of the phospholipids and the hydroxyl groups of the AFP may prevent the phospholipid heads from approaching each other, resulting in a reduction of the vitrification phase transition temperature of the phospholipids, and maintaining the cell membrane fluidity^5,33,36. Since rsfAFP is rich in hydroxyl groups, it can be speculated that the change in the characteristic band of egg yolk lecithin may be due to the formation of hydrogen bonds between the hydroxyl groups in the rsfAFP and the phospholipid head groups.

In situ observation of S. thermophilus and ice crystals via Cryo-TEM

In order to further investigate the mechanism of action of rsfAFP on S. thermophilus cells, we conducted experiments with isolated S. thermophilus cells incubated with or without rsfAFP. These S. thermophilus cells were observed using Cryo-TEM. As shown in Fig. 7, cells without rsfAFP showed significant ruptures of the cell walls, shrinkage, and cellular deformations. In addition, a large number of ice crystals and vesicle-like particles were observed everywhere inside and outside the cells in the PBS control group, whereas the easily recognizable cytoskeleton and typical cell structure were clearly observed in the 0.1 mg/mL rsfAFP-treated cells. These results were consistent with those observed by SEM. Furthermore, the content in ice crystals around the cells treated with rsfAFP was obviously decreased, and the cytoderm in the rsfAFP group was significantly thicker than in the control group, giving the impression that a layer of material was wrapped around the cytoderm. Therefore, we speculated that rsfAFP acted by reducing the formation of ice crystals, therefore limiting the mechanical damage of ice crystals caused to cells. On the other hand, rsfAFP may act as a cellular scaffold wrapped around the outer layer of S. thermophilus cells, thus reducing the mechanical damage caused by ice crystals under freezing stress.

The blurred background represents the ice crystal (green arrow); the cell wall of S. thermophilus ruptured (red arrow); the cell membrane shrinkage (purple arrow); the rsfAFP wrapped around S. thermophilus (yellow arrows); vesicles formed by bacterial rupture (orange arrows). The concentration of rsfAPP used for the cryoprotective measurement was 0.1 mg/mL, and 20 mM PBS was used as a negative control.