Snow flea antifreeze peptide for cryopreservation of lactic acid bacteria

Fresh foods with high water activity undergo chemical reactions and microbial growth that lead to deterioration. Cryopreservation mitigates this but ice crystal growth and recrystallization during freezing and thawing damage cellular and tissue structures, causing quality loss. Common cryoprotectants (skim milk, sugars, phosphates for foods; glycerol, DMSO for cells) do not regulate ice recrystallization and may impart sensory or toxicological drawbacks. Antifreeze proteins (AFPs) exhibit thermal hysteresis activity (THA), ice recrystallization inhibition (IRI), and can modulate ice nucleation, making them promising cryoprotectants. Snow flea AFP, a hyperactive glycine-rich insect AFP with high THA, is a candidate for preserving lactic acid bacteria (LAB). However, large-scale application is limited by low natural yields and high purification costs, and the cryoprotective mechanism on microorganisms remains unclear. This study aims to express a recombinant snow flea antifreeze peptide (rsfAFP) in Bacillus subtilis, evaluate its THA and IRI, assess its cryoprotective effects on Streptococcus thermophilus under freezing stress, and elucidate mechanisms of interaction with ice and bacterial membranes.

- Conventional cryoprotectants lack control over ice recrystallization and may introduce undesirable properties (sweetness, high salt/calorie) or cytotoxicity (e.g., glycerol, DMSO). - AFPs from various organisms display THA and IRI, regulating ice growth and providing cryoprotection; insect AFPs are often hyperactive. - Snow flea AFP has a polyproline II helical bundle structure with high THA (1–2 orders of magnitude above most plant/fish AFPs) and potential as a cryoprotectant for LAB. Prior work focused on ice interaction mechanisms rather than microbial cryoprotection. - Heterologous expression in bacteria is used to overcome yield and cost barriers; Bacillus subtilis is food-safe, but expression of hyperactive snow flea AFP in B. subtilis had not been reported. - AFPs’ Janus mechanism involves ice-binding and non-ice-binding faces; hydrogen bonding to phospholipid headgroups can maintain membrane fluidity during freezing.

- Construction and expression: rsfAFP comprises two repeats of a 6.5 kDa snow flea AFP (175 aa). The gene was codon-optimized for B. subtilis WB800N, synthesized, double-digested with BamHI/XbaI, cloned into pHT43 (pHT43-SF-P), and transformed into B. subtilis WB800N by electroporation. Positive clones were confirmed by PCR (543 bp band). Expression optimization identified 30 °C, 4 h, and 1 mM IPTG as optimal. Proteins from culture medium and extracts were analyzed by Tricine-SDS-PAGE and western blot. - Purification and identification: His-tag affinity purification on Ni2+ resin with stepwise imidazole washes and elutions (100–400 mM). Purity >95% by SDS-PAGE. The excised band underwent in-gel tryptic digestion and Nano LC-MS/MS; Mascot analysis indicated 90.86% sequence coverage. - Thermal hysteresis activity (THA): Differential scanning calorimetry (DSC; PerkinElmer) measured freezing/melting transitions at different holding temperatures; compared rsfAFP with bovine serum albumin (BSA) control. - Ice recrystallization inhibition (IRI): Polarized light microscopy with a Linkam cold stage. Samples (3 µL TBS ± 0.1 mg/mL rsfAFP) were rapidly cooled to −50 °C, held 5 min, warmed to −14 °C, held, then cycled between −14 and −12 °C (1 °C/min) for 5 cycles with imaging to assess crystal growth. - Single ice crystal morphology: Nanoliter osmometer in silicone oil; 0.5 nL of 0.1 mg/mL rsfAFP was frozen to −20 °C, then warmed to Tm, and cooled to Tf; temperature control ±0.01 °C. Growth rate and morphology recorded via CCD. - Cryoprotective assessment on S. thermophilus: Cells grown in M17 to OD600 ~1.0 (~10^8 CFU/mL), washed in PBS. Test formulations: 0.1 mg/mL rsfAFP; positive controls 15% glycerol (v/v), 1.0 mg/mL sucrose, 1.0 mg/mL skim milk; negative control 20 mM PBS. Aliquots (0.3 mL) were frozen at −20 °C for 24 h and subjected to two freeze–thaw cycles (2 h interval). Outcomes: survival rate by rapid OD600 method (post/pre-freeze ×100), metabolic activity (INT-based assay), acidifying activity (pH over time), and growth under prolonged freezing (−20 °C up to 14 days) measured by OD600. - Scanning electron microscopy (SEM): Freeze-dried cells sputter-coated with gold; imaged to assess morphology after freeze–thaw with/without cryoprotectants. - FTIR spectroscopy: Interactions with membrane mimetic assessed using egg yolk lecithin. Samples (lecithin, rsfAFP, and mixture) prepared in KBr (1:100) and analyzed; characteristic headgroup bands monitored for shifts. - Cryo-TEM: Post-freezing cell suspensions (±0.1 mg/mL rsfAFP) vitrified (Vitrobot) on carbon grids and imaged on Talos F200C G2 under low-dose conditions to visualize cells and surrounding ice. - Statistics: SPSS 17.0; data as mean ± SD from ≥3–4 independent experiments; Duncan’s multiple range test; significance at P < 0.05.

- Expression and identification: rsfAFP was successfully expressed in B. subtilis WB800N, purified to >95% purity, and confirmed by Nano LC-MS/MS with 90.86% sequence coverage. - Antifreeze activities: Compared with BSA, rsfAFP exhibited a clear thermal hysteresis exothermic peak and significantly higher THA at the same holding temperatures. rsfAFP addition significantly reduced ice content at given TH conditions. - IRI: After five freeze–thaw cycles, control samples showed large, rounded recrystallized ice, whereas rsfAFP (0.1 mg/mL) maintained significantly smaller ice crystals, indicating strong IRI activity. - Ice morphology modulation: In ultrapure water, single ice crystals rapidly filled the field as flat disks; with 0.1 mg/mL rsfAFP, ice growth was markedly slowed and crystals assumed a hexagonal morphology, consistent with binding to the prism plane. - Cryoprotection of S. thermophilus (−20 °C, 24 h, 2 freeze–thaw cycles): • Survival rate increased from 8.86% (PBS) to 93.21% with 0.1 mg/mL rsfAFP, outperforming 1.0 mg/mL sucrose (60.71%), 1.0 mg/mL skim milk (65.39%), and 15% glycerol (79.80%). • Metabolic activity (% of pre-freeze): PBS 28.34%, sucrose 50.04%, skim milk 62.65%, glycerol 71.06%, rsfAFP 82.54%. • Acid production: rsfAFP-treated cells showed significantly higher acidifying activity than commercial cryoprotectants, consistent with improved metabolism. • Freezing stability (−20 °C storage): After 14 days, survival decreased by 97.88% (PBS), 61.57% (glycerol), and only 38.50% (rsfAFP). - SEM: Without cryoprotectants, cells showed severe structural damage, rupture, and content leakage; sucrose and glycerol partially protected; rsfAFP preserved intact, rounded cell morphology with minimal damage, indicating reduced mechanical stress from large ice crystals. - FTIR: Lecithin headgroup peaks (e.g., ~1241.6 cm−1 P=O and ~969.95 cm−1 C–C–N+) shifted in the presence of rsfAFP, consistent with hydrogen bonding between rsfAFP hydroxyl groups and phospholipid headgroups, stabilizing the membrane. - Cryo-TEM: Controls displayed ruptured cell walls, shrinkage, abundant ice crystals and vesicle-like particles; rsfAFP-treated samples showed recognizable cytoskeleton, thicker apparent cytoderm with rsfAFP wrapping the cell exterior, and reduced surrounding ice, supporting dual mechanisms of ice regulation and membrane protection.

The study demonstrates that rsfAFP provides superior cryoprotection to S. thermophilus compared with common cryoprotectants at much lower concentrations. The data align with the Janus mechanism of antifreeze proteins: rsfAFP’s ice-binding face adsorbs to ice growth interfaces, increasing curvature and interfacial vapor pressure to depress freezing and inhibit recrystallization, while the non-ice-binding face disrupts interfacial water ordering and ice nucleation. Concurrently, FTIR evidence suggests rsfAFP forms hydrogen bonds with phospholipid headgroups, maintaining membrane fluidity during freezing and mitigating dehydration-induced gel-phase transitions. Cryo-TEM corroborates reduced ice formation around cells and an rsfAFP layer wrapping the cell exterior, which likely dampens mechanical stresses from ice crystals. Together, these mechanisms explain the observed improvements in survival, metabolic activity, acid production, and long-term freezing stability. Because rsfAFP is a macromolecule that does not penetrate cells, it may offer a safer alternative to small organic solvents like glycerol, with promising applicability in food and biopreservation contexts.

This work reports, for the first time, the successful expression of recombinant snow flea antifreeze peptide (rsfAFP) in Bacillus subtilis and establishes its significant thermal hysteresis and ice recrystallization inhibition activities. rsfAFP modulates single-crystal ice morphology and growth, and markedly enhances the survival, metabolic activity, acid production, and freezing stability of Streptococcus thermophilus under freezing stress, outperforming conventional cryoprotectants. Mechanistically, rsfAFP both regulates ice nucleation/growth and interacts with membrane phospholipid headgroups via hydrogen bonding, preserving membrane integrity. These synergistic actions underpin its cryoprotective efficacy. The findings highlight rsfAFP as a promising, low-toxicity cryoprotectant for lactic acid bacteria and frozen foods. Future work should elucidate the detailed molecular interactions of rsfAFP with specific ice planes and membrane components, assess scalability and cost-effective production, and expand testing across diverse microorganisms and food matrices.

The precise molecular mechanism by which rsfAFP regulates ice at the atomic level remains unclear. The study focuses on a single bacterial species (S. thermophilus), and broader applicability across organisms and product systems was not evaluated. Quantitative comparisons of THA against a broader panel of AFPs were not provided.