Cryopreservation is crucial for food storage, but ice crystal growth and recrystallization damage food quality and cell-based foods like probiotics. Current antifreeze agents like skim milk, sugars, phosphates, glycerol, and DMSO have limitations in regulating ice crystal formation during recrystallization or thawing, leading to mechanical stress damage. Antifreeze proteins (AFPs) offer promise due to their thermal hysteresis activity (THA), ice recrystallization inhibition (IRI), and ice nucleation isomerization abilities. However, obtaining AFPs from natural sources is costly and yields are low. A glycine-rich AFP from Canadian snow fleas exhibits high THA, making it a potential cryoprotectant. This study aimed to express and produce a recombinant snow flea antifreeze peptide (rsfAFP) in *B. subtilis*, investigate its effects on ice crystal morphology, THA, and IRI, and determine its cryoprotective effect on *S. thermophilus* under freezing stress. The study also aims to explore the mechanism of rsfAFP interaction with cells and ice.

The literature review highlights the challenges of current cryopreservation methods for food and cells, emphasizing the limitations of conventional antifreeze agents in preventing ice crystal damage. It emphasizes the potential of antifreeze proteins (AFPs) as a superior alternative due to their unique properties, including thermal hysteresis activity (THA), ice recrystallization inhibition (IRI), and ice nucleation isomerization. The review also discusses previous research on snow flea AFPs, focusing on their interaction with ice crystals while noting a lack of studies on their protective effects on microorganisms. The review points out the advantages of using *B. subtilis* as an expression system for heterologous proteins due to its non-pathogenic nature and suitability for food and medical applications.

The study involved constructing a recombinant *B. subtilis* WB800N strain expressing rsfAFP. The rsfAFP gene was synthesized, cloned into the pHT43 vector (creating pHT43-SF-P), and transformed into *B. subtilis*. Expression was optimized, and the protein was purified using affinity chromatography. The identity and purity of rsfAFP were confirmed using PCR, Western blot, and Nano LC-MS/MS. Antifreeze activity was assessed by measuring THA using differential scanning calorimetry (DSC) and IRI using polarized light microscopy and a nanoliter osmometer. Cryoprotective effects on *S. thermophilus* were evaluated by assessing survival rate, metabolic activity, acid production, and growth stability after freeze-thaw cycles. Cell morphology was examined using scanning electron microscopy (SEM), while the interaction between rsfAFP and the cell membrane was investigated using Fourier transform infrared (FTIR) spectroscopy with egg yolk lecithin as a model. Cryo-TEM was used for in situ observation of *S. thermophilus* and ice crystals in the frozen state. Statistical analysis was performed using SPSS 17.0.

rsfAFP was successfully expressed, purified, and identified in *B. subtilis*. It exhibited significant THA and IRI ability, effectively inhibiting ice crystal growth and recrystallization. rsfAFP significantly improved the survival rate (from 8.86% to 93.21%), metabolic activity, acid production, and freezing stability of *S. thermophilus* compared to control groups using sucrose, skim milk, and glycerol. SEM images showed that rsfAFP maintained the integrity of *S. thermophilus* cells under freezing stress, unlike the control groups. FTIR analysis indicated that rsfAFP interacts with the phospholipid head groups of lecithin, likely through hydrogen bonding. Cryo-TEM revealed that rsfAFP inhibits ice crystal formation around *S. thermophilus* cells and forms a protective layer around the cells.

The findings demonstrate that rsfAFP is a highly effective cryoprotectant for *S. thermophilus*, surpassing the performance of traditional cryoprotectants. The mechanism appears to involve a dual action: inhibiting ice crystal growth by binding to the ice interface (Janus effect) and protecting the cell membrane by interacting with phospholipids through hydrogen bonding. The large molecular weight of rsfAFP prevents it from penetrating the cell membrane, unlike small organic molecules such as glycerol, suggesting a safer cryoprotective agent. The study's results support the use of rsfAFP as a novel and effective cryoprotectant in food processing and preservation.

This study successfully produced and characterized a recombinant snow flea antifreeze peptide (rsfAFP) with significant cryoprotective properties. rsfAFP effectively protects *S. thermophilus* from freezing damage by inhibiting ice crystal formation and interacting with the cell membrane. This research highlights the potential of rsfAFP as a safe and efficient cryoprotectant for lactic acid bacteria and other frozen foods. Future research could focus on optimizing rsfAFP production, investigating its effectiveness on other microorganisms, and exploring its applications in diverse food preservation systems.

The study focused on *S. thermophilus* as a model organism. Further research is needed to assess the effectiveness of rsfAFP on other lactic acid bacteria and other types of cells. While the interaction between rsfAFP and lecithin provides insight into its interaction with cell membranes, further investigation is needed to fully understand the precise molecular mechanisms involved in membrane protection. The study used a specific freezing protocol, and the effectiveness of rsfAFP might differ under varying freezing conditions.