The relationship between the antibacterial effect of Nisin and its storage time

As a natural antimicrobial peptide, Nisin is widely used in food preservation, pharmaceuticals, and other fields due to its high-efficiency inhibition of Gram-positive bacteria (e.g., Listeria monocytogenes, Staphylococcus aureus) and its GRAS (Generally Recognized as Safe) certification. However, its antimicrobial efficacy is not constant and gradually attenuates with prolonged storage. During storage, environmental factors (temperature, humidity, oxygen, light) and carrier characteristics (e.g., food matrix, packaging materials) act together to cause damage to Nisin’s molecular structure, inactivation of active sites, and ultimately a reduction in its antibacterial ability. Starting from the mechanisms of Nisin activity attenuation, this article systematically analyzes the law of how storage time affects its antimicrobial efficacy, key influencing factors, and activity preservation strategies, providing theoretical support for the efficient application of Nisin.

I. Core Mechanisms of Nisin’s Antimicrobial Efficacy Attenuation with Storage Time

Nisin’s antimicrobial activity relies on its specific spatial structure—it contains 5 lanthionine rings (formed by cross-linking of cysteine and dehydroalanine), which are key active sites for its binding to microbial cell membranes and inhibition of cell wall synthesis. The attenuation of antimicrobial efficacy due to prolonged storage essentially stems from the destruction of Nisin’s molecular structure or inactivation of its active sites, with specific mechanisms categorized into three types:

(I) Oxidative Degradation: Destruction of Thioether Bonds and Disulfide Bonds in the Molecule

Nisin molecules contain multiple thioether bonds (the core structure of lanthionine rings) and a small number of disulfide bonds. Oxygen in the storage environment (especially free oxygen) undergoes oxidation reactions with these chemical bonds, leading to the breakage of ring structures and unfolding of molecular chains. For example, oxygen oxidizes thioether bonds into sulfoxide or sulfone derivatives, causing lanthionine rings to lose spatial stability and fail to bind to receptors on microbial cell membranes. Meanwhile, oxidation also converts amino groups (-NH₂) in Nisin molecules into imino groups (-NH-) or carbonyl groups (-CO-), further disrupting molecular polarity and reducing its electrostatic adsorption capacity with cell membranes. Studies have shown that after 30 days of storage in an oxygen-rich environment, the breakage rate of thioether bonds in Nisin can reach 35%, the corresponding minimum inhibitory concentration (MIC) increases from the initial 0.5 μg/mL to 2.0 μg/mL, and antimicrobial activity decreases by 75%.

(II) Hydrolysis Reaction: Peptide Bond Breakage Leading to Molecular Fragmentation

Nisin is a polypeptide composed of 34 amino acids. Moisture in the storage environment (especially free water) acts as a medium to trigger hydrolysis of peptide bonds—water molecules attack peptide bonds (-CO-NH-), breaking them into small peptide fragments or free amino acids. These fragmented products lose their intact spatial structure, cannot form active sites that interact with microorganisms, and completely lose their antibacterial ability. For example, under high humidity (relative humidity > 80%), after 60 days of storage, the peptide bond hydrolysis rate of Nisin reaches 40%, the proportion of intact Nisin molecules in the solution decreases from the initial 95% to 45%, and the diameter of the inhibition zone against Staphylococcus aureus shrinks from 15 mm to 8 mm (smaller inhibition zones indicate weaker antimicrobial efficacy). In addition, proteases in food matrices (e.g., milk protease in milk, cathepsin in meat) can also accelerate Nisin hydrolysis, further shortening its activity retention time.

(III) Aggregation Inactivation: Intermolecular Interactions Causing Active Site Shielding

During storage, Nisin molecules tend to aggregate due to hydrophobic interactions and electrostatic attraction—hydrophobic amino acid residues (e.g., alanine, leucine) approach each other to form a hydrophobic core, leading to molecular chain entanglement. Meanwhile, in neutral or weakly alkaline environments, Nisin molecules carry negative charges; if metal ions (e.g., Ca²⁺, Mg²⁺) are present, they promote molecular aggregation through electrostatic bridging. The resulting Nisin aggregates shield its active sites (e.g., lanthionine rings), preventing contact with microbial cell membranes. Furthermore, aggregates may precipitate, further reducing the concentration of free active Nisin in the system. For example, after 20 days of storage of Nisin in a food matrix containing 0.1 mol/L Ca²⁺, the aggregation rate reaches 50%, the concentration of active Nisin in the supernatant decreases from the initial 100 μg/mL to 30 μg/mL, and the inhibition rate against Listeria drops from 98% to 60%.

II. Law of How Storage Time Affects Nisin’s Antimicrobial Efficacy and Key Influencing Factors

The attenuation of Nisin’s antimicrobial efficacy with storage time is not linear but follows a pattern of "slow attenuation in the early stage – rapid attenuation in the middle stage – stabilization in the later stage". It is regulated by storage environment (temperature, humidity, oxygen, light), carrier characteristics (food matrix, packaging materials), and initial concentration, with significant differences in attenuation rates under different conditions.

(I) Attenuation Law: Staged Changes in Activity

1. Early Stage (0–15 days): Slow Attenuation Stage

In the early stage of storage, environmental factors (e.g., oxygen, moisture) have not yet significantly acted on Nisin molecules, and only a small number of active sites are damaged, resulting in slow attenuation of antimicrobial efficacy. For example, after 15 days of storage of Nisin at 4°C, low humidity (relative humidity < 30%), and oxygen-free conditions, the antimicrobial activity retention rate remains above 85%, and the inhibition rate against target bacteria decreases by less than 10%. During this stage, Nisin molecules exist in monomer form, with active sites basically intact, enabling them to exert antibacterial effects efficiently.

2. Middle Stage (15–60 days): Rapid Attenuation Stage

As storage time extends, the continuous action of oxygen and moisture intensifies Nisin’s oxidative degradation and hydrolysis, increases molecular aggregation rate, and inactivates a large number of active sites, leading to a rapid decline in antimicrobial efficacy. For example, after 60 days of storage of Nisin at 25°C, high humidity (relative humidity > 70%), and oxygen-rich conditions, the antimicrobial activity retention rate drops sharply from 85% to 30%, the MIC value increases by 3–4 times, and the inhibition rate against target bacteria decreases from 90% to 40%. During this stage, the proportion of intact Nisin molecules is less than 50%, and the system is dominated by degraded fragments and aggregates, resulting in a significant reduction in antibacterial ability.

3. Later Stage (60+ days): Stabilization Stage

In the later stage of storage, most active Nisin has undergone degradation or aggregation, and the attenuation rate of the remaining small amount of active molecules slows down, with antimicrobial efficacy tending to stabilize (but activity has been significantly reduced). For example, after 90 days of storage of Nisin at 37°C, oxygen-rich, and high-humidity conditions, the antimicrobial activity retention rate stabilizes at approximately 20%; further extending the storage time to 120 days, the retention rate decreases by only 5%. At this stage, there are almost no intact active molecules in the system, and the antibacterial effect is weak.

(II) Key Influencing Factors: Joint Regulation of Attenuation Rate by Environment and Carriers

1. Temperature: Accelerates Oxidation and Hydrolysis Reactions

Temperature is a core factor affecting Nisin activity attenuation—high temperatures significantly increase molecular movement rate and accelerate the rate constants of oxidative degradation and hydrolysis reactions. For example, after 60 days of storage at 4°C, Nisin’s activity retention rate reaches 65%; while under the same storage time at 37°C, the activity retention rate is only 25%, and the attenuation rate increases by 2.6 times. Low temperatures (< 4°C) can reduce molecular kinetic energy, slow down chemical bond breakage and molecular aggregation, and significantly extend Nisin’s activity retention time. Therefore, refrigeration (4°C) or freezing (-18°C) is the preferred storage condition for Nisin.

2. Humidity: Promotes Hydrolysis and Aggregation

Moisture is the medium for Nisin’s hydrolysis reaction—high-humidity environments increase the content of free water in the system, accelerating peptide bond breakage. Meanwhile, moisture also reduces the threshold of hydrophobic interactions between Nisin molecules, promoting aggregation. For example, after 30 days of storage at 90% relative humidity, Nisin’s hydrolysis rate reaches 35% and aggregation rate reaches 40%; while under the same storage time at 10% relative humidity, the hydrolysis rate is only 8% and aggregation rate is only 15%. Therefore, storage at low humidity (relative humidity < 30%) can effectively reduce hydrolysis and aggregation, maintaining Nisin activity.

3. Oxygen: Dominates Oxidative Degradation

Oxygen is the main cause of oxidation of Nisin’s thioether bonds and disulfide bonds—oxygen-rich environments significantly accelerate the destruction of active sites. For example, after 60 days of storage under oxygen-free conditions, Nisin’s activity retention rate reaches 55%; while under the same storage time in oxygen-rich conditions, the activity retention rate is only 30%, and oxidative degradation contributes to more than 60% of activity loss. In addition, light (especially ultraviolet light) can activate oxygen to generate reactive oxygen species (e.g., hydroxyl radicals ·OH), further intensifying oxidation reactions. Therefore, Nisin must be stored in the dark.

4. Food Matrix: Affects Stability and Solubility

The pH and components (e.g., proteins, fats, metal ions) of the food matrix change the state of Nisin:

pH: Nisin has the best stability in acidic environments (pH 2.0–5.0) (molecules exist in stable monomer form), while in alkaline environments (pH > 7.0), it is prone to deamination reactions, leading to rapid activity decline. For example, after 30 days of storage of Nisin in fruit juice at pH 3.0, the activity retention rate reaches 70%; while under the same storage time in dairy products at pH 8.0, the activity retention rate is only 40%.

Metal ions: Divalent metal ions such as Ca²⁺ and Mg²⁺ promote Nisin aggregation through electrostatic interactions, while chelating agents such as EDTA (ethylenediaminetetraacetic acid) can bind to metal ions to reduce aggregation. For example, in a Nisin solution with 0.05% EDTA added, the aggregation rate after 30 days of storage decreases from 40% to 15%, and the activity retention rate increases by 25%.

Proteins: Proteins in food (e.g., casein in milk) can form complexes with Nisin, reducing antimicrobial efficacy by shielding active sites, but they can also reduce Nisin hydrolysis (proteins adsorb moisture, lowering free water content). The balance of these effects depends on the specific matrix.

5. Packaging Materials: Key to Blocking Environmental Factors

The oxygen resistance, moisture resistance, and light resistance of packaging materials directly determine the microenvironment of Nisin:

Oxygen resistance: Aluminum foil composite films and EVOH (ethylene-vinyl alcohol copolymer) films have excellent oxygen resistance, which can increase Nisin’s activity retention rate by more than 30% after 60 days of storage compared to ordinary PE films.

Moisture resistance: Polyvinylidene chloride (PVDC) films have strong moisture resistance, which can reduce moisture entry and decrease Nisin’s hydrolysis rate by 20%–30%.

Light resistance: Brown PET films can block ultraviolet light, avoid the generation of reactive oxygen species, and reduce Nisin’s oxidative degradation rate by 15%–20%.

III. Storage Optimization Strategies to Extend Nisin’s Antimicrobial Efficacy

To address Nisin’s activity attenuation caused by storage time, efforts should be made from three aspects: "controlling environmental factors, optimizing carrier characteristics, and improving formulations" to slow down the attenuation rate and maximize the duration of antimicrobial efficacy.

(I) Environmental Regulation: Creating Storage Conditions with Low Oxygen, Low Temperature, Low Humidity, and Darkness

1. Temperature Control

Priority should be given to refrigerated (4°C) or frozen (-18°C) storage. If room-temperature storage is required, the temperature should be controlled below 25°C to avoid accelerated degradation due to high temperatures. For example, when storing Nisin at room temperature (25°C), air conditioning or constant-temperature warehouses should be used to ensure the temperature fluctuation range is < ±5°C.

2. Humidity Control

Moisture-proof packaging (e.g., aluminum-plastic composite bags containing desiccants) should be used to control the relative humidity of the storage environment at 30%–50%, avoiding hydrolysis caused by high humidity. For example, adding silica gel desiccants to Nisin powder packaging can maintain the relative humidity inside the bag below 20%, increasing the activity retention rate by 20% after 60 days of storage.

3. Oxygen Isolation and Light Protection

Oxygen-resistant packaging materials (e.g., aluminum foil bags, EVOH composite films) should be used, and vacuum or nitrogen-filled packaging should be adopted to reduce oxygen contact. Meanwhile, opaque or brown packaging should be selected to avoid light-induced oxidation reactions. For example, Nisin solutions packaged in brown glass bottles with nitrogen filling have an activity retention rate 35% higher than those in transparent glass bottles after 30 days of storage.

(II) Carrier Optimization: Improving the Compatibility of Food Matrices and Packaging Materials

1. pH Adjustment

A small amount of organic acids (e.g., citric acid, lactic acid) should be added to alkaline food matrices (e.g., neutral dairy products) to adjust the pH to 5.0–6.0, improving Nisin stability. For example, adding 0.1% citric acid to milk increases Nisin’s activity retention rate from 40% to 65% after 30 days of storage without affecting milk flavor.

2. Chelating Agent Addition

EDTA (addition amount: 0.02%–0.05%) should be added to foods containing metal ions (e.g., mineral water, soy products) to chelate Ca²⁺ and Mg²⁺, reducing Nisin aggregation. For example, adding 0.03% EDTA to soy products decreases Nisin’s aggregation rate from 50% to 20% after 20 days of storage and increases antimicrobial efficacy by 30%.

3. Packaging Material Upgrade

High-barrier packaging should be selected based on food type. For example, cooked meat products use "PET/AL/EVOH/PE" multi-layer composite films, which synergistically improve oxygen resistance, moisture resistance, and light resistance, enabling Nisin to have an activity retention rate of over 55% after 60 days of storage (compared to only 30% for ordinary PE films).

(III) Formulation Improvement: Enhancing Nisin’s Own Stability

1. Microencapsulation

Edible wall materials (e.g., maltodextrin, gum arabic, chitosan) should be used to microencapsulate Nisin (particle size: 1–10 μm). The wall materials can block direct contact between oxygen/moisture and Nisin, while controlling the slow release of Nisin to extend antimicrobial duration. For example, Nisin microcapsules encapsulated with gum arabic have an activity retention rate of 60% after 60 days of storage at 25°C and high humidity (compared to only 25% for the unencapsulated group), and can achieve "rapid initial release (for bacteriostasis) + slow subsequent release (for long-term preservation)" in food.

2. Compound Antimicrobial Agents

Nisin should be compounded with other natural antimicrobial agents (e.g., plant essential oils, lysozyme). The synergistic effect compensates for the insufficient bacteriostasis caused by Nisin’s activity attenuation. For example, the compound of Nisin (0.1%) and carvacrol (0.05%) increases the inhibition rate against E. coli from 30% (Nisin alone) to 75% after 30 days of storage. Since carvacrol’s antimicrobial activity is less affected by storage time, it can synergistically maintain overall bacteriostatic efficacy.

3. Powderization Treatment

The stability of Nisin powder is significantly higher than that of Nisin solution (powder has low free water content, resulting in slow oxidation and hydrolysis rates). Therefore, powder form should be preferred for storage, and dissolution should be performed before use. For example, Nisin powder has an activity retention rate of 70% after 180 days of storage at 4°C and low humidity; while Nisin solution has an activity retention rate of only 30% under the same storage conditions.

IV. Conclusions and Future Directions

The antimicrobial efficacy of Nisin exhibits an attenuation pattern of "slow in the early stage – rapid in the middle stage – stable in the later stage" as storage time extends. The core causes lie in oxidative degradation, hydrolysis reactions, and aggregation inactivation, which damage its active structure. The storage environment (temperature, humidity, oxygen, light) and carrier characteristics (food matrix, packaging materials) are key factors regulating the attenuation rate: low temperature, low humidity, oxygen isolation, light protection, and acidic food matrices can significantly delay attenuation; high-barrier packaging materials and microencapsulation technology can effectively improve Nisin’s storage stability.

Future research should focus on three aspects:

1. Accurate Prediction of Attenuation Models

Combine molecular dynamics simulations and accelerated aging experiments to establish mathematical models for Nisin activity attenuation under different environments and carriers, enabling accurate prediction of antimicrobial efficacy.

2. Intelligent Responsive Storage Technology

Develop environment-responsive packaging (e.g., humidity-responsive microcapsules, oxygen-responsive coatings) that automatically releases protective agents (e.g., antioxidants, chelating agents) when environmental factors exceed standards, maintaining Nisin activity in real time.

3. Genetically Engineered Modified Nisin

Modify Streptococcus lactis through genetic engineering to synthesize Nisin derivatives with more stable structures (e.g., mutants with enhanced thioether bond stability), improving degradation resistance at the molecular level and extending antimicrobial efficacy after storage.

Through the above strategies, the activity retention time of Nisin can be further extended, and its natural antimicrobial advantages can be fully exerted, providing a more efficient solution for the food industry to reduce the use of chemical preservatives and ensure food safety.