The core of Nisin's freeze-drying technology

Nisin’s freeze-drying technology, through the synergy of low-temperature dehydration and protectants, can significantly extend its shelf life (up to 2–3 years at room temperature) while maximizing retention of antimicrobial activity. The core lies in optimizing freeze-drying process parameters and protectant formulations to minimize ice crystal damage and degradation of active ingredients.

I. Core Principles of Extending Shelf Life and Preserving Activity via Freeze-Drying

1. Key Logic for Extending Shelf Life

Freeze-drying removes moisture from the Nisin system through three steps—"freezing, sublimation, desorption"—reducing moisture content to below 5%. The low-moisture environment inhibits microbial growth, reduces oxidation and enzymatic hydrolysis, and prevents Nisin structure damage, fundamentally extending the storage cycle. Compared to room-temperature drying (shelf life of 3–6 months), freeze-dried Nisin has a 4–6 times longer shelf life under sealed room-temperature conditions.

2. Core Mechanism for Preserving Activity

The entire freeze-drying process is conducted at low temperatures (-40℃~-10℃), avoiding Nisin peptide chain breakage and thioether ring decomposition caused by high heat. Meanwhile, sublimation drying eliminates liquid water, reducing damage to Nisin’s active sites (e.g., key structures binding to lipid Ⅱ) by water molecules. This results in over 90% retention of Nisin’s antimicrobial activity after freeze-drying.

II. Optimization of Key Freeze-Drying Process Parameters

1. Pre-Freezing Stage: Minimizing Ice Crystal Damage

Pre-Freezing Temperature: Controlled at -40℃~-50℃. Rapid freezing forms fine ice crystals in the Nisin solution, avoiding structural damage to Nisin molecules by large ice crystals and improving activity retention by 15%–20%.

Pre-Freezing Time: 2–4 hours to ensure complete freezing of the solution (no residual liquid water), preventing local collapse during sublimation.

2. Sublimation Drying Stage: Efficient Dehydration with Activity Preservation

Vacuum Degree: Maintained at 10–30 Pa. The low-vacuum environment accelerates ice crystal sublimation, shortens drying time (8–12 hours), and reduces oxidation risks.

Shelf Temperature: Gradually increased to 0℃~10℃ to avoid thermal denaturation of Nisin structure due to sudden temperature rises while ensuring full ice crystal sublimation.

3. Desorption Drying Stage: Removing Residual Moisture

Temperature Control: Increased to 20℃~30℃ to desorb residual moisture adsorbed on Nisin surfaces, resulting in a final moisture content of ≤3%.

Time Optimization: 4–6 hours. Excessive moisture shortens shelf life, while insufficient moisture may cause Nisin powder to absorb moisture and agglomerate.

III. Protectant Formulation Optimization: Enhancing Activity Retention

1. Core Protectant Types and Functions

Sugar Protectants: Sucrose, trehalose (5%–10% addition). They bind to Nisin via hydrogen bonds, replacing water molecules to maintain its spatial structure. Trehalose outperforms sucrose, improving activity retention by 10%–15%.

Protein/Amino Acid Protectants: Bovine Serum Albumin (BSA, 1%–2%), glycine (2%–3%). They reduce Nisin aggregation during freeze-drying and inhibit oxidation.

Polymer Protectants: Polyethylene Glycol (PEG 6000, 1%–2%), hydroxypropyl-β-cyclodextrin (2%–4%). They improve Nisin’s solubility and dispersibility, preventing powder agglomeration after freeze-drying.

2. Example of Composite Protectant Formulation (100mL Nisin Solution)

1g Nisin + 6g trehalose + 2g glycine + 1g PEG 6000. After freeze-drying, activity retention exceeds 95%, and over 85% antimicrobial activity is maintained after 2 years of room-temperature storage.

IV. Storage and Application Precautions for Freeze-Dried Products

1. Optimized Storage Conditions

Packaging: Use aluminum foil vacuum bags with desiccants to isolate oxygen and moisture, avoiding moisture absorption and degradation.

Environment: Store at 0℃~25℃, avoiding high temperatures, direct strong light, and humid environments (which accelerate activity loss).

2. Application Adaptation Key Points

Reconstitution: Rapidly dissolve with sterile water or buffer (pH 5.0–7.0), avoiding prolonged stirring that may damage Nisin structure.

Compatibility: After reconstitution, avoid mixing with strong acid, strong alkali, or high-salt systems to prevent activity reduction. It can be used synergistically with other biological preservatives (e.g., ε-polylysine) to enhance antimicrobial efficacy.

V. Common Problems and Solutions

Severe Activity Loss: Optimize pre-freezing temperature (lower to -50℃), increase trehalose addition, and extend desorption drying time.

Powder Agglomeration: Reduce polymer protectant dosage, seal packaging immediately after freeze-drying, and control storage humidity ≤60%.

Shortened Shelf Life: Check final moisture content (must be ≤3%), enhance packaging tightness, and avoid bag damage during transportation.