The stability of Nisin under liposome embedding

As a cationic antimicrobial peptide, the activity of Nisin is susceptible to external factors such as protease hydrolysis, pH fluctuations, temperature changes, and ionic strength, which limits its long-acting applications in the food and biomedicine fields. Liposomes, as biomimetic membrane vesicles composed of phospholipid bilayers, can significantly enhance the stability of Nisin and prolong its active half-life through three mechanisms: physical barrier protection, microenvironment regulation, and structure-adaptive binding. The specific mechanisms are as follows:

I. Physical Barrier Protection Mechanism of Phospholipid Bilayers

The core function of liposomes is to isolate Nisin from the adverse external environment through their vesicle structure, fundamentally reducing activity loss, which serves as the basis for stability enhancement.

Spatial Isolation Blocks Degradation PathwaysDuring liposomal encapsulation, water-soluble Nisin is mainly entrapped in the internal aqueous phase of vesicles, while a small amount of hydrophobic fragments can be embedded in the hydrophobic region of the phospholipid bilayer. This encapsulated structure forms a physical barrier that blocks the contact between Nisin and external degrading enzymes such as trypsin and pepsin, preventing peptide chain cleavage caused by enzymatic hydrolysis. Meanwhile, it isolates Nisin from metal ions and oxidants (e.g., hydrogen peroxide) in food matrices or body fluids, avoiding oxidative damage to disulfide bonds in the Nisin molecule and maintaining its antimicrobial active conformation. Experimental data show that after incubation in simulated gastric fluid for 2 hours, the residual activity rate of free Nisin is less than 30%, whereas that of liposome-encapsulated Nisin can reach over 80%.

Reduces Direct Impact of Environmental FactorsExternal factors such as pH fluctuations, high temperature, and light exposure can directly affect the molecular conformation of Nisin (e.g., destruction of α-helical structures), leading to loss of antimicrobial activity. The phospholipid bilayer of liposomes possesses a certain degree of elasticity and buffering capacity:

In acidic environments, protonation of phosphate groups in phospholipid molecules enhances membrane rigidity, reducing hydrogen ion penetration to the internal Nisin. In alkaline environments, dissociation of amino groups in phospholipids maintains pH stability in the internal microenvironment of vesicles.

During high-temperature processing (e.g., food pasteurization), the ordered arrangement of the liposomal membrane can reduce the impact of thermal motion on Nisin. Moreover, cholesterol-modified liposomal membranes exhibit stronger thermal stability, capable of maintaining vesicle integrity at 60–80°C and protecting Nisin from thermal denaturation.

II. Microenvironment Regulation Mechanism of Liposomes

The interior of liposomes can form a local microenvironment distinct from the outside, which further enhances Nisin stability by regulating parameters such as charge distribution and water activity to meet the activity requirements of Nisin.

Charge Adaptation Reduces Molecular AggregationNisin is a cationic polypeptide (with an isoelectric point of approximately 8.8), and the phospholipid bilayer of liposomes can be regulated by selecting phospholipids with different charge properties:

When liposomes are prepared using negatively charged phospholipids (e.g., phosphatidylglycerol, phosphatidylserine), the negative charges on the inner wall of vesicles can form electrostatic adsorption with cationic residues (e.g., lysine, arginine) of Nisin, dispersing Nisin uniformly in the internal aqueous phase and preventing active site masking caused by molecular aggregation.

This electrostatic interaction can also fix the active conformation of Nisin, preventing the loss of antimicrobial activity due to molecular folding and deformation (the α-helical structure of Nisin is critical for binding to bacterial cell membranes).

Water Activity Regulation Delays DegradationNisin is prone to hydrolysis in high water activity environments. The water activity of the internal aqueous phase of liposomes can be regulated through the hydrophobic effect of phospholipids—the hydrophobic tails of the phospholipid bilayer restrict the free movement of water molecules, reducing the water activity (aw) of the internal aqueous phase and slowing down the rate of non-enzymatic hydrolysis of Nisin. Meanwhile, the liposomal membrane can prevent excessive infiltration of external water, maintaining the dryness of the internal microenvironment, which is particularly suitable for applications in high-moisture foods (e.g., beverages, dairy products).

III. Synergistic Mechanism of Liposome Structural Adaptability for Enhanced Efficacy

Through component modification or structural optimization of liposomes, their protective capacity for Nisin can be further enhanced, improving the durability of stability.

Membrane Component Modification Enhances Barrier Stability

Cholesterol Addition: Cholesterol can embed in the hydrophobic region of the phospholipid bilayer, filling the gaps between phospholipid molecules, enhancing membrane rigidity and compactness, reducing Nisin leakage, improving the temperature and pH tolerance of liposomes, and extending the stability cycle of the encapsulated system.

Saturated Phospholipid Selection: Saturated phospholipids (e.g., dipalmitoylphosphatidylcholine, DPPC) have fatty acid chains without double bonds, exhibiting tighter molecular arrangement and superior membrane structural stability compared to unsaturated phospholipids, thus providing more durable protection for Nisin.

Surface Polyethylene Glycol (PEG) Modification: Grafting PEG chains onto the liposome surface can form a steric hindrance layer. On the one hand, it reduces the aggregation and fusion of liposomes in food or body fluids, maintaining vesicle integrity. On the other hand, it blocks non-specific binding between external proteins and liposomes, reducing indirect loss of Nisin.

Particle Size Optimization Improves Encapsulation StabilityThe particle size of liposomes directly affects encapsulation efficiency and stability. Small-sized liposomes (< 200 nm) have a large specific surface area, higher encapsulation efficiency, better dispersibility in the system, and are less prone to sedimentation and aggregation. Meanwhile, small-sized liposomes have greater membrane curvature and stronger intermolecular forces between phospholipids, resulting in more stable vesicle structures and reduced Nisin leakage loss. In contrast, large-sized liposomes tend to cause Nisin release due to gravitational sedimentation or membrane rupture, exhibiting poor stability.

IV. Synergistic Effects of Stability Enhancement and Application Verification

The stability protection of Nisin by liposomes is not the result of a single mechanism, but a synergy of physical barrier, microenvironment regulation, and structural adaptation, which is ultimately reflected in the following aspects:

Improved Storage Stability: After 3 months of storage at room temperature, the residual activity rate of liposome-encapsulated Nisin can still reach over 75%, while that of free Nisin is less than 40%.

Improved Processing Stability: During food processing procedures such as high-temperature sterilization and pH adjustment, the activity loss of liposome-encapsulated Nisin is reduced by 50%–60%.

Improved Biological Stability: In the in vivo digestive tract environment, liposomes can protect Nisin from degradation by gastric acid and proteases, improving its bioavailability.

V. Key Factors Affecting Stability

Phospholipid Type and Ratio: Negatively charged phospholipids have better encapsulation stability than neutral phospholipids, and the optimal effect is achieved when the cholesterol addition amount is controlled at 20%–30% (mass ratio relative to phospholipids).

Encapsulation Process: The reverse-phase evaporation method has a higher encapsulation efficiency than the thin-film hydration method, and the prepared liposomes provide better protection for Nisin.

External Environment: Liposome-encapsulated Nisin exhibits higher stability in neutral or weakly alkaline environments. Excessively strong acidity (pH < 3) can still cause membrane structure damage, requiring the combined use of pH regulators.