The molecular structure and stability of Nisin

Nisin is a cyclic polypeptide containing lanthionine, and its stability is significantly affected by factors such as pH, temperature, and food matrix. Application stability can be enhanced through structural modification or formulation optimization. Detailed analysis is as follows:

I. Molecular Structural Characteristics of Nisin

1. Basic Structural Framework

Nisin is a cationic polypeptide composed of 34 amino acid residues with a molecular weight of approximately 3510 Da. It forms 5 thioether bonds (lanthionine and β-methyllanthionine) within the molecule, constructing 3 cyclic structures (Ring A, Ring B, Ring C). Core amino acids include alanine, leucine, and isoleucine, among others. It contains 2 lysine residues and 1 histidine residue, giving the molecule an overall positive charge (isoelectric point, pI ≈ 8.5).

2. Key Functional Structures

Thioether Ring Structures: The core of its antimicrobial activity, capable of inserting into bacterial cell membranes to form pores and disrupt osmotic balance.

Amino-Terminal and Carboxy-Terminal: The hydrophobic region of the amino-terminal binds to bacterial cell membranes, while the cyclic structure of the carboxy-terminal stabilizes pore morphology. Both synergistically ensure antimicrobial activity.

Modified Amino Acids: The molecule contains 3 lanthionine residues, 2 β-methyllanthionine residues, and 1 dehydroalanine residue. These modified structures enhance molecular rigidity and antimicrobial specificity.

II. Stability of Nisin and Influencing Factors

1. Impact of pH

Acidic Environment (pH 2.0~5.0): Optimal stability, with the longest half-life at pH 3.0 (up to several months at 25℃). Suitable for acidic foods (e.g., yogurt, fruit juice, pickled products).

Neutral to Alkaline Environment (pH ≥6.0): Molecular conformation is prone to change, and thioether bonds undergo hydrolysis and cleavage, leading to rapid loss of antimicrobial activity. At pH 7.0 and 25℃, over 50% of activity is lost within 1 week.

2. Impact of Temperature

Low to Medium Temperature (≤60℃): Over 80% of activity is retained after short-term treatment (e.g., pasteurization). It can remain stable for more than 12 months when stored at refrigeration temperature (4℃).

High Temperature Treatment (>100℃): High temperatures accelerate peptide bond cleavage and conformational damage. Over 90% of activity is lost after autoclaving at 121℃ for 15 minutes. However, high-temperature stability improves under acidic conditions (pH 3.0), with approximately 60% of activity retained after treatment at 100℃ for 10 minutes.

3. Impact of Food Matrix

Proteins and Fats: Proteins (e.g., whey protein) and fats in food can adsorb Nisin, reducing its free concentration and indirectly affecting stability and antimicrobial efficacy. The addition amount needs to be appropriately increased.

Metal Ions: Divalent metal ions such as Ca²⁺ and Mg²⁺ can bind to Nisin, enhancing its structural stability. Heavy metal ions such as Fe³⁺ and Cu²⁺ catalyze oxidation reactions, accelerating Nisin degradation.

Enzymes: Proteases in food (e.g., trypsin, pepsin) hydrolyze Nisin’s peptide bonds, reducing its activity. Prolonged storage in high-protein hydrolysis systems should be avoided.

4. Other Influencing Factors

Light: Ultraviolet light damages Nisin’s thioether bonds and peptide bonds, leading to activity loss. Opaque packaging should be used for storage.

Oxygen: Oxidation reactions damage the molecular structure. Vacuum or nitrogen-filled packaging can extend its shelf life.

III. Optimization Strategies to Enhance Nisin Stability

1. Structural Modification and Modification

Chemical Modification: Acetylation or PEGylation of Nisin’s amino-terminal enhances its stability in neutral and alkaline environments, while reducing sensitivity to protease hydrolysis.

Genetic Engineering Modification: Site-directed mutagenesis replaces easily hydrolyzable amino acid residues, improving high-temperature stability and broad-spectrum antimicrobial activity.

2. Microencapsulation Embedding

Nisin microcapsules are prepared using wall materials such as sodium alginate, β-cyclodextrin, and chitosan through spray drying or emulsification-coacervation methods.Embedding isolates oxygen, proteases, and metal ions, increasing activity retention by 30%~50% in neutral foods and high-temperature processed foods.

3. Synergistic Stabilization through Compound Formulation

Compound with EDTA: EDTA chelates heavy metal ions to reduce oxidative degradation, while enhancing Nisin’s antimicrobial activity against Gram-negative bacteria.

Synergy with Other Preservatives: Compound formulation with potassium sorbate or ε-polylysine not only improves stability but also expands the antimicrobial spectrum and reduces the addition amount of a single preservative.

4. Application Process Optimization

Addition Under Acidic Conditions: In the later stage of food processing (e.g., cooling phase), adjust the system pH to 4.0~5.0 before adding Nisin to reduce the impact of high temperature and alkaline environments.

Low-Temperature Storage and Transportation: Control the product storage temperature at 0~4℃, avoid light and high-temperature environments, and extend the shelf life.