Influencing factors of Nisin's antibacterial activity and its optimization strategies

As a high-efficiency natural bacteriostatic agent targeting Gram-positive bacteria (G⁺), Nisin is widely applied in food, pharmaceutical, and feed industries due to its advantages of high safety, no residue, and biodegradability. Its bacteriostatic activity relies on the integrity of its molecular structure and effective binding to target sites (bacterial cell membranes). However, during production, storage, and application, Nisin is susceptible to multiple factors such as environmental conditions, substrate characteristics, and microbial resistance, leading to activity attenuation or inactivation. This article systematically analyzes the core factors influencing Nisin's bacteriostatic activity, reveals the regulatory laws of activity at the molecular mechanism level, and proposes targeted optimization strategies. Combined with application practice verification, it provides theoretical support and technical reference for the efficient utilization of Nisin.

I. Core Factors Influencing Nisin's Bacteriostatic Activity

(1) Environmental Physicochemical Factors

1. pH Value

Nisin's molecular structure consists of 34 amino acid residues, including 5 lanthionine (Lan) and 2 methyllanthionine (MeLan) special structures. Its bacteriostatic activity is closely related to molecular charge:

Optimal pH Range: At pH 2.0~6.0, Nisin molecules carry a positive charge, facilitating binding to negatively charged components (e.g., phosphatidylglycerol, lipoteichoic acid) on G⁺ bacterial cell membranes, forming transmembrane pores that cause intracellular substance leakage and cell death.

Activity Attenuation Mechanism: At pH＞6.5, Nisin molecules undergo conformational changes, resulting in reduced positive charge and weakened electrostatic attraction to cell membranes. Meanwhile, the molecules are prone to degradation by proteases. At pH＜2.0, the strong acidic environment may destroy the lanthionine ring structure, leading to molecular denaturation and inactivation.

Example: At neutral pH 7.0, the minimum inhibitory concentration (MIC) of Nisin against Staphylococcus aureus is 3~5 times higher than that at pH 4.0; at pH 8.0, activity loss exceeds 60%.

2. Temperature

Temperature regulates Nisin's bacteriostatic activity by affecting molecular stability and bacterial cell membrane fluidity:

Thermal Stability Characteristics: Under acidic conditions (pH 2.0~5.0), Nisin exhibits strong thermal stability, retaining over 70% activity after sterilization at 121℃ for 15 min. Under neutral or alkaline conditions, high temperatures (>80℃) accelerate molecular conformational damage, leading to rapid activity attenuation.

Temperature Adaptability: Nisin activity can be maintained for a long time during refrigerated (4℃) or room temperature (25℃) storage. During heat processing (e.g., canned food sterilization), temperature and time must be controlled to avoid excessive thermal damage.

Example: At pH 6.0, heating at 85℃ for 30 min results in 50% loss of Nisin's bacteriostatic activity; while at pH 3.0, activity loss is only 15% under the same conditions.

3. Ionic Strength and Metal Ions

The ion concentration and metal ion types in the environment significantly affect the binding efficiency between Nisin and bacterial cell membranes:

Inhibition by High Ionic Strength: High concentrations of Na⁺, Cl⁻, Ca²⁺, etc., in food or culture media can shield the electrostatic interaction between Nisin and cell membranes, reducing molecular penetration ability. Especially when NaCl concentration exceeds 5%, bacteriostatic activity decreases by 40%~60%.

Dual Effects of Metal Ions:

Inhibitory Ions: Divalent ions such as Mg²⁺ and Mn²⁺ can bind to bacterial cell membrane phospholipids, enhancing membrane stability and hindering Nisin pore formation.

Synergistic Ions: Zn²⁺, Cu²⁺, etc., can bind to amino and carboxyl groups in Nisin molecules, improving molecular structure stability and enhancing cell membrane damage. Low concentrations (0.01~0.1 mmol/L) of Zn²⁺ can increase Nisin's bacteriostatic activity by 2~3 times.

4. Water Activity (Aw)

Water activity affects Nisin molecular diffusion and bacterial metabolic activity:

Optimal Aw Range: At Aw 0.90~0.98, Nisin molecules have strong diffusion ability, enabling rapid contact with bacteria and exerting effects.

Activity Limitation Mechanism: At Aw＜0.85, Nisin molecular fluidity and diffusion rate decrease, while bacterial metabolic activity is inhibited, weakening bacteriostatic effects. At Aw＞0.98, the high-moisture environment easily promotes Nisin degradation, especially under neutral or alkaline conditions.

(2) Substrate and Application System Factors

1. Food/Culture Medium Composition

Macromolecular components in complex matrices affect Nisin activity through adsorption, encapsulation, and other mechanisms:

Interference by Proteins and Fats: Proteins such as casein and myoglobin in milk and meat products can form complexes with Nisin, and fat droplets can encapsulate Nisin molecules, hindering their binding to bacterial cell membranes. For example, in meat products containing 10% fat, the MIC value of Nisin is 2~4 times higher than that in fat-free systems.

Influence of Carbohydrates: High concentrations of carbohydrates such as sucrose and starch reduce water activity, indirectly affecting Nisin diffusion, but have weak direct inhibitory effects on activity.

Natural Inhibitors: Some foods contain proteases (e.g., trypsin in milk, cathepsin in meat) that can degrade Nisin's peptide bonds, leading to activity loss. Polyphenols in beer and fruit juices can interact with Nisin through hydrophobic interactions, reducing its bacteriostatic activity.

2. Microorganism-Related Factors

(1) Bacterial Species and Characteristics

Strain Specificity: Nisin exhibits significant bacteriostatic effects against G⁺ bacteria (e.g., Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis) with MIC values typically ranging from 0.01~5 μg/mL. Due to the outer membrane barrier of Gram-negative bacteria (G⁻), its bacteriostatic activity is extremely weak and requires combination with EDTA or other chelating agents.

Bacterial Growth Stages: Bacteria in the logarithmic growth phase have high cell membrane permeability and are sensitive to Nisin. Bacteria in the stationary phase form mature cell walls, resulting in reduced bacteriostatic effects. Spores are highly tolerant to Nisin and require combination with high-temperature treatment.

(2) Resistance Mechanisms

Long-term use of Nisin may induce bacterial resistance through the following main mechanisms:

Reduced synthesis of lipoteichoic acid (LTA) on bacterial cell membranes, decreasing binding sites for Nisin.

Bacteria secrete Nisin-degrading enzymes (e.g., Nisinase) that hydrolyze the lanthionine ring in Nisin molecules.

Upregulated expression of multidrug resistance pumps (e.g., ABC transporters) on bacterial cell membranes, expelling Nisin from cells.

(3) Nisin's Intrinsic Characteristics

1. Purity and Molecular Weight

Industrial Nisin products typically have a purity of 2.5%~50%. Impurities (e.g., culture medium components, miscellaneous proteins) reduce the effective concentration and affect bacteriostatic effects. High-purity (>90%) Nisin exhibits more stable bacteriostatic activity and requires lower dosage.Nisin has a molecular weight of approximately 3510 Da. The intact molecular structure is the basis for its bacteriostatic activity, and degradation products (e.g., fragmented peptide chains) have no bacteriostatic effect.

2. Concentration and Action Time

Bacteriostatic activity is concentration-dependent. Within a certain range (0.1~10 μg/mL), higher concentrations result in stronger bacteriostatic effects. However, beyond the critical concentration, the activity improvement slows down, and high concentrations may accelerate bacterial resistance development.Prolonged action time enhances bacteriostatic effects. Typically, Nisin needs to contact bacteria for more than 1~4 hours to completely inhibit bacterial growth and reproduction.

II. Optimization Strategies for Nisin's Bacteriostatic Activity

(1) Environmental Condition Optimization

1. pH Regulation

Application System pH Adjustment: Adjust the pH of food or culture media to 4.0~5.5. For example, add organic acids such as lactic acid and citric acid to meat products, or utilize the intrinsic acidity of raw materials (e.g., fruit juice pH 3.0~4.5) in beverages to enhance Nisin activity.

Composite Acidity Regulators: Use a 1:1 ratio of lactic acid and citric acid. This not only adjusts pH but also synergizes with Nisin for bacteriostasis, improving effects by over 30% compared to single organic acids.

2. Temperature and Processing Technology Adaptation

Low-Temperature Storage and Processing: Store Nisin products in sealed refrigeration (4℃) to avoid high-temperature exposure. In food processing, prioritize low-temperature sterilization processes (e.g., pasteurization at 63℃/30 min) or add Nisin during the cooling stage after sterilization to reduce thermal damage.

Short-Time High-Temperature Treatment: For high-temperature processing (e.g., canned food sterilization), adopt a "high-temperature short-time" process (135℃/2~5 s) under acidic conditions to minimize Nisin activity loss.

3. Ionic Environment Optimization

Salt Concentration Control: Reduce NaCl usage (≤3%) in food processing or replace part of NaCl with low-sodium salt (KCl) to reduce the inhibitory effect of ionic strength on Nisin.

Addition of Synergistic Metal Ions: Add 0.05~0.1 mmol/L ZnSO₄ or CuCl₂ to the application system to synergize with Nisin and enhance bacteriostatic activity. Avoid simultaneous use with high concentrations of inhibitory ions such as Mg²⁺ and Mn²⁺.

4. Water Activity Regulation

In food processing, maintain Aw within the optimal range of 0.90~0.98 by controlling moisture content (e.g., meat product Aw 0.92~0.95) and adding appropriate humectants (e.g., glycerol, propylene glycol) to ensure Nisin molecular diffusion efficiency.

(2) Application System Optimization

1. Matrix Component Regulation

Reduce Interfering Components: In high-protein and high-fat foods, remove some impurities through centrifugation, filtration, or other processes, or appropriately increase Nisin dosage (50%~100% higher than in ordinary foods).

Add Protease Inhibitors: In protease-containing foods (e.g., milk, meat), add soybean trypsin inhibitor (STI) or EDTA (0.05%~0.1%) to inhibit Nisin degradation.

Select Suitable Matrices: Prioritize applying Nisin in acidic, low-fat, and low-protein foods (e.g., fruit juices, beverages, bread) for better bacteriostatic effects.

2. Construction of Composite Bacteriostatic Systems

Composite bacteriostasis is a core strategy to enhance Nisin activity and expand its antibacterial spectrum, achieving a "1+1＞2" effect through synergy:

(1) Compound with Other Natural Bacteriostatic Agents

Plant Extracts: Compound with eugenol, cinnamaldehyde, tea polyphenols, etc. Plant extracts disrupt cell membranes, enhancing Nisin's inhibition of G⁺ bacteria and expanding activity to some G⁻ bacteria (e.g., Salmonella). For example, Nisin (5 μg/mL) + eugenol (0.1 mg/mL) increases the inhibition zone diameter against Escherichia coli by more than 2 times compared to single Nisin.

Biopreservatives: Compound with ε-polylysine and lysozyme to synergistically destroy bacterial cell membranes and cell walls, improving inhibition effects against resistant strains.

(2) Compound with Chemical Bacteriostatic Agents

Chelating Agents: EDTA (0.05%~0.2%) can chelate Ca²⁺ and Mg²⁺ on G⁻ bacterial cell membranes, disrupting the outer membrane structure and allowing Nisin to enter cells and exert effects.

Organic Acids: Lactic acid, citric acid, etc., not only adjust pH but also enhance bacterial cell membrane permeability, synergizing with Nisin for bacteriostasis.

(3) Combination with Physical Technologies

High-Pressure Processing (HPP): High pressure of 300~500 MPa disrupts bacterial cell membrane structure, reducing Nisin's MIC value. For example, HPP (400 MPa/5 min) + Nisin (2 μg/mL) exhibits the same bacteriostatic effect against Listeria monocytogenes as single Nisin (8 μg/mL).

Ultrasound: 20~40 kHz ultrasound promotes Nisin molecular diffusion and destroys bacterial cell walls, improving bacteriostatic efficiency.

Irradiation: Low-dose γ-irradiation (1~3 kGy) enhances bacterial sensitivity to Nisin and synergistically inhibits spore germination.

(3) Optimization of Nisin's Intrinsic Performance

1. Purity Improvement and Formulation Technology

Purification Process Optimization: Adopt gel filtration chromatography, reverse-phase high-performance liquid chromatography (RP-HPLC), and other technologies to increase Nisin purity to over 90% and reduce impurity interference.

Development of Novel Formulations:

Microencapsulation: Use sodium alginate, chitosan, β-cyclodextrin, and other wall materials to prepare Nisin microcapsules (particle size 1~10 μm) through spray drying or emulsification. This protects Nisin from protease degradation and environmental factors, extends shelf life, and achieves sustained release.

Liposome Encapsulation: Encapsulate Nisin in liposomes (particle size 50~200 nm) to enhance penetration ability through G⁻ bacterial outer membranes and expand the antibacterial spectrum.

Composite Film Formulations: Combine Nisin with edible films (e.g., gelatin, pectin films) for food surface preservation, reducing Nisin loss and increasing local concentration.

2. Structural Modification and Derivative Development

Improve Nisin's stability and bacteriostatic activity through molecular modification:

Chemical Modification: Acetylation, PEGylation, and other modifications of amino groups in Nisin molecules reduce degradation rate in neutral/alkaline environments and enhance water solubility and biocompatibility.

Genetic Engineering Modification: Use site-directed mutagenesis (e.g., replacing key amino acid residues in the molecule) to enhance Nisin's protease tolerance and cell membrane binding ability. For example, replacing isoleucine at position 27 of Nisin A with leucine increases its tolerance to Nisinase by 3 times.

(4) Resistance Prevention and Control Strategies

1. Rational Control of Dosage and Frequency

Adopt the "minimum effective concentration" principle, accurately controlling Nisin dosage (typically 0.1~1 μg/mL) based on food type and target bacteria to avoid excessive use.Alternate use of different types of bacteriostatic agents (e.g., rotation of Nisin with plant extracts and chemical bacteriostatic agents) reduces bacterial resistance pressure to a single agent.

2. Combined Inhibition of Resistance Mechanisms

Add resistance pump inhibitors (e.g., reserpine) to block bacterial excretion of Nisin.Combine with cell wall synthesis inhibitors (e.g., penicillin) to destroy bacterial cell wall integrity and reduce the probability of resistance development.

III. Application Practice and Effect Verification

(1) Typical Application Cases

1. Food Preservation Field

Meat Product Preservation: Add Nisin (0.5 μg/g) + lactic acid (0.2%) + EDTA (0.1%) to sausage processing, adjusting the product pH to 5.0. During storage at 4℃, the lag phase of Staphylococcus aureus and Salmonella growth extends from 2 days to 7 days, doubling the shelf life.

Dairy Product Preservation: Add Nisin (1 μg/mL) + lysozyme (0.5 mg/mL) to pasteurized milk. The shelf life at 4℃ extends from 7 days to 14 days without off-odors.

Fruit Juice Preservation: Add Nisin (0.3 μg/mL) + cinnamaldehyde (0.05 mg/mL) to orange juice (pH 3.5). During room temperature storage, mold and yeast growth are significantly inhibited, extending the shelf life by 5~7 days.

2. Food Processing Field

Canned Foods: Adopt the process of "sterilization at 121℃/10 min + Nisin (0.8 μg/g) + citric acid (0.3%)" in mushroom can processing. Compared with traditional processes, the sterilization time is shortened by 5 min, the product color and flavor are improved, and the spore killing rate reaches 99.9%.

Bread Preservation: Add Nisin (0.2 μg/g) + tea polyphenols (0.1%) to bread making. During room temperature storage, the lag phase of mold growth extends from 3 days to 8 days, and the bread hardness increase rate decreases by 40%.

3. Feed and Pharmaceutical Fields

Feed Additives: Add Nisin (50 μg/g) + zinc oxide (0.1%) to piglet feed. This significantly inhibits Bacillus subtilis and Escherichia coli in feed, reduces piglet diarrhea rate by 30%, and increases daily weight gain by 15%.

Pharmaceutical Field: Combined use of Nisin (10 μg/mL) + vancomycin (2 μg/mL) increases bacteriostatic activity against vancomycin-resistant Staphylococcus aureus (VRSA) by 4 times compared to single vancomycin, providing a new scheme for the treatment of drug-resistant bacterial infections.

(2) Effect Evaluation Methods

1. Quantitative Detection of Bacteriostatic Activity

Agar Diffusion Method: Determine the inhibition zone diameter; larger diameters indicate stronger bacteriostatic activity. Suitable for rapid screening of optimization schemes.

Microbroth Dilution Method: Determine the MIC value; smaller MIC values indicate stronger bacteriostatic activity. Used for precise quantification of Nisin's bacteriostatic effects.

Growth Curve Method: Real-time monitor changes in bacterial OD₆₀₀ values using a microplate reader, analyze the effects of Nisin on the bacterial lag phase and logarithmic phase, and evaluate bacteriostatic persistence.

2. Stability Evaluation

Accelerated Aging Test: Store Nisin at 40℃ and 75% RH for 30 days, regularly detecting activity to evaluate stability.

Processing Stability Test: Simulate food processing technologies (e.g., heating, acid-base treatment) and detect Nisin activity retention rate before and after treatment.

3. Practical Application Effect Evaluation

Sensory Evaluation: Assess changes in food color, flavor, and texture after Nisin addition.

Microbial Counting: Regularly detect the total colony count and target bacterial count (e.g., pathogenic bacteria, spoilage bacteria) in food.

Shelf Life Determination: Record the deterioration time of food under specified storage conditions to evaluate preservation effects.

IV. Challenges and Future Development Directions

(1) Existing Challenges

1. Limited Antibacterial Spectrum

Nisin has weak inhibitory effects on G⁻ bacteria and fungi, limiting its application in some foods (e.g., aquatic products, fruits, and vegetables).

2. Insufficient Stability

Nisin is prone to degradation in neutral/alkaline environments, high-temperature processing, and complex matrices, requiring formulation technology to improve stability.

3. Resistance Risk

Long-term and widespread use may lead to the spread of G⁺ bacterial resistance, affecting its sustainable application.

4. High Cost

The complex production process of high-purity Nisin results in higher costs than chemical bacteriostatic agents, limiting large-scale application.

(2) Future Development Directions

1. Innovation in Antibacterial Spectrum Expansion Technology

Develop composite systems of Nisin with novel chelating agents and nanomaterials (e.g., silver nanoparticles) to enhance inhibitory effects against G⁻ bacteria and fungi.Construct novel Nisin analogs with activity against G⁺ bacteria, G⁻ bacteria, and fungi through genetic engineering modification.

2. Research and Development of High-Efficiency Formulation Technologies

Develop intelligent responsive microcapsule formulations (e.g., pH-responsive, temperature-responsive) to achieve precise release of Nisin in target environments.Utilize nanotechnology (e.g., nanofibers, nanoemulsions) to improve Nisin's stability and targeting.

3. Optimization of Low-Cost Production Processes

Optimize fermentation processes (e.g., breeding high-yield strains, optimizing culture medium composition) to increase Nisin fermentation yield.Develop efficient and low-cost purification processes to reduce the production cost of high-purity Nisin.

4. Expansion of Multi-Field Applications

Expand Nisin's application in the pharmaceutical field (e.g., skin infections, oral care) and develop novel antibacterial formulations.Apply Nisin as a feed additive and in the agricultural field to replace some antibiotics, supporting green breeding and sustainable agricultural development.

Nisin's bacteriostatic activity is regulated by multiple factors, including environmental physicochemical conditions, application system characteristics, intrinsic structure, and microbial resistance. The core influence mechanisms focus on the regulation of molecular structure stability and cell membrane binding efficiency. Through strategies such as pH and ionic environment optimization, composite bacteriostatic system construction, formulation technology upgrading, and resistance prevention and control, Nisin's bacteriostatic activity, stability, and application scope can be significantly improved. In practical applications, it is necessary to accurately match optimization schemes according to specific scenarios (e.g., food type, processing technology, target bacteria) to achieve efficient utilization of Nisin. In the future, with the innovative development of molecular biology and formulation technology, its application prospects in food, pharmaceutical, feed, and other fields will be broader, providing important support for the industrialization of natural bacteriostatic agents.