The antibacterial activity of Nisin is temperature-dependent

I. Structural Characteristics and Antibacterial Mechanism Basis of Nisin

Nisin, a natural polypeptide antibiotic secreted by Lactococcus lactis, consists of 34 amino acid residues with five thioether bonds (lanthionine and β-methyllanthionine) forming a unique rigid cyclic structure. Its antibacterial mechanism primarily involves disrupting bacterial cell membrane integrity:

Binding to lipid II on the Gram-positive bacterial membrane to inhibit peptidoglycan synthesis;

Inserting into the membrane to form pores, causing leakage of intracellular ions and ATP, leading to bacterial death.

The influence of temperature on Nisin's spatial structure, membrane interaction, and stability represents the core mechanism of its temperature-dependent antibacterial activity.

II. Low-Temperature Environment (≤25℃): Activity Maintenance and Membrane Fluidity Regulation

1. Balance of Structural Stability and Bacteriostatic Efficiency

Nisin's lanthionine ring structure remains stable at low temperatures, resisting denaturation. Studies show no significant change in the minimum inhibitory concentration (MIC) of Nisin against Staphylococcus aureus after storage at 4℃ for 1 month.

Low temperature reduces bacterial membrane fluidity, affecting Nisin's binding efficiency to lipid II: when membrane lipids are in the gel phase, the energy barrier for Nisin insertion increases, potentially requiring longer action time for bacteriostasis, but the final bacteriostatic rate still maintains >80% (e.g., treating Listeria at 10℃ for 24 hours).

2. Application Advantages in Cold-Chain Foods

In low-temperature dairy products (e.g., refrigerated yogurt), Nisin extends shelf life by inhibiting psychrophilic bacteria (e.g., Pseudomonas). For example, at 4℃, 0.1 μg/mL Nisin keeps Bacillus cereus count in fresh milk ≤10³ CFU/mL within 7 days, while the control group doubles.

III. Moderate Temperature Range (25–60℃): Activity Peak and Kinetic Optimization

1. Temperature Sensitivity of Membrane Interaction

At 25–40℃, bacterial membranes are in the liquid crystal phase with moderate fluidity, optimizing Nisin's binding rate to lipid II and membrane pore formation efficiency. For instance, the bacteriostatic rate constant (k) of Nisin against Bacillus subtilis at 37℃ is 1.8 times that at 25℃, shortening bacterial death time from 60 to 35 minutes.

Increasing temperature promotes exposure of Nisin's hydrophobic regions, enhancing hydrophobic interactions with membrane lipids. Experiments show Nisin inserts into S. aureus membranes 3 times faster at 45℃ than at 25℃, reducing membrane potential depolarization time from 15 to 5 minutes.

2. Synergistic Effect with Thermal Sterilization

During pasteurization (60℃, 30 min), Nisin enhances sterilization via:

High temperature denaturing bacterial membrane proteins, facilitating Nisin binding to exposed lipid II;

Increased conformational flexibility of Nisin at 50–60℃, with α-helix content in the N-terminal region (responsible for pore formation) increasing by 12%, improving membrane penetration. For example, 0.5 μg/mL Nisin at 60℃ reduces Bacillus stearothermophilus spore survival rate from 20% to 3%.

IV. High-Temperature Environment (>60℃): Activity attenuation and Structural Denaturation

1. Thermal Stability Limit and Molecular Structure Damage

Nisin's thermal stability is pH-dependent: it retains 70% activity after 80℃ for 30 min under acidic conditions (pH 2.0), but loses 50% activity at neutral pH (pH 7.0) after 60℃ for 10 min. This occurs because high temperature hydrolyzes lanthionine bonds, opening the cyclic structure and losing lipid II binding ability.

Differential scanning calorimetry (DSC) shows Nisin's thermal denaturation temperature (Td) is ~75℃ at pH 6.0. Above this, its β-sheet structure collapses rapidly, with antibacterial activity decreasing exponentially. For example, after 100℃ for 5 min, Nisin's MIC against Listeria increases from 0.25 to 2.0 μg/mL.

2. Application Limitations in High-Temperature Food Processing

Nisin is almost completely inactivated during ultra-high temperature sterilization (UHT, 135℃, 2 s) or canning (121℃, 15 min), requiring post-sterilization addition. However, in acidic cans (e.g., fruit cans with pH <4.5), Nisin partially retains activity during 60–80℃ sterilization, synergistically inhibiting thermophilic bacteria (e.g., Clostridium botulinum).

V. Influence of Temperature on Nisin's Action Spectrum

1. Differential Temperature Responses of Gram-Positive Bacteria

For thermophiles (e.g., Streptococcus thermophilus), Nisin's optimal bacteriostatic temperature coincides with their growth optimum (45–50℃), when membrane lipids have high unsaturated fatty acid content, facilitating Nisin insertion.

For mesophiles (e.g., S. aureus), Nisin is most active at 37℃, requiring higher concentrations for equivalent bacteriostasis at low temperatures (10℃).

2. Weak Temperature Dependence in Gram-Negative Bacteria

Nisin is naturally insensitive to Gram-negative bacteria due to the outer membrane lipopolysaccharide barrier, but high temperatures (50–60℃) transiently disrupt outer membrane integrity, allowing Nisin to penetrate to the inner membrane. For example, Nisin's bacteriostatic rate against E. coli increases from 10% at room temperature to 35% at 55℃, still significantly lower than for Gram-positive bacteria.

VI. Synergistic Model of Temperature-Time-Concentration

Nisin's antibacterial activity can be described by the Arrhenius equation: the relationship between antibacterial rate constant (k) and temperature (T) is k = A・e^(-Ea/RT), where the activation energy (Ea) is ~40 kJ/mol at 25–40℃ and rises to 65 kJ/mol above 60℃, indicating higher energy barriers for activity attenuation at high temperatures. In practice, a "temperature-effective action time" curve can be established:

Example: In pH 4.5 juice with 100 IU/mL Nisin:

Bacteriostatic activity lasts 14 days at 25℃;

Effective time shortens to 7 days at 40℃;

Only maintains 24 hours at 60℃.

VII. Temperature Regulation Strategies in Practical Applications

1. Optimization Schemes for Food Preservation

Refrigerated foods (4–8℃): Low-concentration Nisin (50–100 IU/mL) inhibits spoilage bacteria while avoiding nutrient damage from high temperatures.

Pasteurized products (60–70℃): Nisin synergizes with heat to reduce sterilization temperature or time. For example, 70℃ for 10 min with Nisin enhances sterilization by 40% compared to 80℃ for 15 min alone, while preserving more vitamin C.

2. Temperature Control in Industrial Production

Fermentation: The optimal temperature for L. lactis to produce Nisin is 30–35℃; exceeding 40℃ inactivates synthetic enzymes, decreasing yield.

Formulation preparation: In spray drying, inlet temperature should be controlled below 180℃, and adding protectants (e.g., lactose) increases Nisin activity retention from 60% to 85%.

Conclusion

Nisin's antibacterial activity exhibits a non-linear relationship with temperature: long-acting bacteriostasis at low temperatures via structural stability, activity peaks in moderate temperatures through membrane interaction kinetics, and activity attenuation at high temperatures due to structural denaturation. This temperature dependence grants unique advantages in cold-chain foods and pasteurization, while high-temperature processing requires combined pH regulation or composite preservation technologies. Deep understanding of temperature's influence on Nisin's structure-activity relationship provides a theoretical basis for developing thermostable Nisin derivatives (e.g., site-directed mutagenesis of lanthionine bonds) and precise temperature-controlled preservation technologies.