Nisin's freeze-drying technology

I. Mechanism of Freeze-Drying on Nisin Activity

As a polypeptide with 34 amino acids, Nisin's activity relies on the rigid structure formed by 5 intramolecular disulfide bonds. Activity loss during freeze-drying mainly stems from:

Mechanical Damage by Ice Crystals: Ice crystals (5–50 μm diameter) formed during water freezing physically squeeze Nisin molecules, causing disulfide bond cleavage (e.g., disruption of Cys²⁰-Cys²⁷ bonding).

Solute Concentration Effect: The concentration of the unfrozen solute phase increases during freezing, enhancing hydrophobic interactions between Nisin molecules and triggering aggregation (forming aggregates ≥100 nm).

Oxidative Stress: Residual oxygen during drying oxidizes polypeptide side chains (e.g., Met¹¹, His²⁴), altering the structure of the antibacterial active center (activity loss rate up to 30%–40%).

Optimized freeze-drying processes can enhance activity retention to >85% through vitrification protection (forming an amorphous glassy matrix for solutes) and microenvironment regulation (reducing ice crystal formation rate).

II. Optimization of Key Freeze-Drying Process Parameters

(1) Pre-Freezing Stage: Ice Crystal Control and Protective Agent Screening

Cooling Rate Regulation:

Slow freezing (0.5–1°C/min) forms large ice crystals (>20 μm diameter), suitable for low-concentration Nisin solutions (<1 mg/mL). Slow crystallization expels water to reduce solute concentration.

Fast freezing (10–20°C/min) forms nano-scale ice crystals (<5 μm diameter), suitable for high-concentration systems (≥5 mg/mL), reducing mechanical damage but requiring protective agents to prevent aggregation.

Protective Agent Formulation Technology:

Excipients: 10%–20% (w/v) trehalose or sucrose bind to hydroxyl groups on the Nisin molecular surface via hydrogen bonds, replacing water molecules to maintain structural stability. The glass transition temperature (Tg’) can increase to -35°C (Tg’ is -45°C without protective agents).

Antioxidants: 0.1%–0.5% (w/v) ascorbyl palmitate scavenges oxygen free radicals generated during freeze-drying, reducing Met oxidation rate from 22% to <5%.

Surfactants: 0.05% (w/v) Tween-80 reduces gas-liquid interfacial tension, inhibiting surface denaturation of Nisin during drying and increasing activity retention by 15%–20%.

(2) Drying Stage: Vacuum Degree and Temperature Gradient Control

Primary Drying (Sublimation Stage):

Maintain vacuum at 10–30 Pa to avoid spray bottle phenomena caused by bubble boiling. Temperature control is phased:

Early stage: -30°C to -20°C, heating at 0.5°C/min to ensure slow ice crystal sublimation, reducing residual moisture to 10%–15%.

Later stage: -20°C to -10°C, increasing heating rate to 1°C/min to promote deep ice crystal release. At this time, Nisin molecules form a "glass-wrapped" structure in the protective agent matrix.

Secondary Drying (Desorption Stage):

Heat to 20–30°C, vacuum <10 Pa, lasting 12–24 hours to reduce moisture content to <3%. Note: When the temperature exceeds 35°C, the trehalose matrix slightly browns, causing 5%–8% Nisin activity loss.

III. Evaluation System and Technological Innovations for Activity Retention

(1) Multi-Dimensional Activity Characterization Methods

Biological Potency Assay: Using a modified turbidity method with Listeria monocytogenes ATCC 19115 as the indicator bacterium, cultivate at 37°C, pH 6.5 for 4 hours. The minimum inhibitory concentration (MIC) of the optimized freeze-dried sample remains at 0.5 IU/mL (consistent with fresh samples).

Structural Integrity Analysis:

Circular Dichroism (CD): The β-sheet characteristic peak intensity of freeze-dried samples at 218 nm retains >92%, proving no significant secondary structure damage.

Mass Spectrometry (MALDI-TOF): Molecular weight detection shows the main peak at 3510 Da (standard 3513 Da), with the oxidation modification peak (+16 Da) ratio <3%.

Thermal Stability Verification: After autoclaving freeze-dried samples at 121°C for 20 minutes, activity retention reaches 78% (only 55% for non-freeze-dried samples), attributed to the buffering effect of the dry matrix against thermal denaturation.

(2) Innovations in New Freeze-Drying Technologies

Combined Vacuum Freeze-Spray Drying: First, spray Nisin solution (10 mg/mL) into microdroplets (50–100 μm diameter), then directly freeze-dry in a -40°C vacuum environment to form a porous microsphere structure (specific surface area ≥10 m²/g). Compared with traditional freeze-drying, activity retention increases from 85% to 91%, and rehydration is significantly improved (completely dissolved within 30 seconds).

Inert Gas-Protected Freeze-Drying: Fill with 99.99% high-purity nitrogen (oxygen content <10 ppm) during pre-freezing and drying stages, combined with a 0.2% (w/v) dithiothreitol (DTT) reducing environment, controlling Met oxidation rate to <1%, suitable for preparing pharmaceutical-grade Nisin (e.g., oral bacteriostats).

Low-Temperature Eutectic Freeze-Drying: Mix Nisin with a 15% (w/v) glycine-proline binary eutectic system, using the eutectic point (-48°C) to lower the ice crystal formation temperature, making the amorphous matrix of the freeze-dried sample more uniform, with activity retention exceeding 95%, but costs increasing by 20%–30% compared to traditional processes.

IV. Challenges and Solutions in Industrial Application

Temperature Uniformity in Scale Production:

Industrial freeze-dryers (volume ≥1000 L) need multi-shelf temperature control (temperature difference ≤±1°C) with a gas circulation system (flow rate 0.5 m/s) to avoid local overheating. Example: A food additive enterprise uses a 12-shelf freeze-dryer with PLC program stage temperature control, causing activity retention fluctuations of ≤3% between batches.

Energy Consumption Optimization and Cost Control:

Introduce heat pump technology to recover cooling capacity during the drying stage, reducing energy consumption by 35% compared to traditional freeze-drying. Meanwhile, adopt the "high-concentration freeze-drying + ultrafine grinding" process, increasing the initial Nisin concentration from 5 mg/mL to 20 mg/mL, and increasing unit energy consumption output by 4 times.

Rehydration Performance Improvement:

Add 0.1% (w/v) cyclodextrin to embed Nisin before freeze-drying, forming a β-cyclodextrin-polypeptide inclusion complex. The freeze-dried powder dissolves 50% faster in water, suitable for instant products like beverages.

V. Frontier Research Directions

Nanoscale Freeze-Drying Protection Strategy: Encapsulate Nisin in trehalose-chitosan nanoparticles (100–200 nm diameter) before freeze-drying. The nano-space confinement effect reduces molecular movement, with activity retention up to 98%, and stability in gastric acid environment (pH 1.2) increased by 3 times.

3D Printing Freeze-Drying Technology: Combine low-temperature extrusion molding with freeze-drying to prepare Nisin drug-loaded scaffolds with gradient pore structures. When used as wound dressings, they enable sustained release for 14 days (release rate 0.5–1.0 IU/cm²·h).

Theoretical Model Prediction: Construct a Nisin-protective agent interaction model based on molecular dynamics simulation (MD) to predict optimal freeze-drying parameters (e.g., optimal cooling rate = 1.2°C/min, disulfide bond energy loss <5%), shortening process development cycles by >50%.