The stability mechanism of Nisin under different pH conditions

The stability of Nisin is highly dependent on the pH value of the solution, and its core mechanism stems from the regulation of molecular charge state, spatial conformation, and peptide bond hydrolysis rate by pH. As a cationic polypeptide composed of 34 amino acids, Nisin contains 5 thioether ring structures (lanthionine and β-methyllanthionine) in its molecule, which are crucial for maintaining its antibacterial activity. Meanwhile, the molecule has a large number of acidic and basic amino acid residues, and the protonation/deprotonation state under different pH conditions directly affects molecular stability. The specific rules and mechanisms are as follows:

I. Strongly Acidic Conditions (pH 2.0–3.0): Optimal Stability and Intact Structure

The strongly acidic environment is the optimal pH range for Nisin stability. Under these conditions, the molecular half-life can reach several months (at 25°C) without significant attenuation of antibacterial activity. The core mechanisms include two aspects:

Stable Charge Distribution Maintains Spatial ConformationUnder strongly acidic conditions, the carboxyl groups (aspartic acid and glutamic acid residues) in the Nisin molecule are fully protonated (-COOH), and the amino groups (lysine and arginine residues) are also fully protonated (-NH₃⁺), giving the molecule an overall strong positive charge. The electrostatic repulsion between positive charges can effectively prevent molecular aggregation. Meanwhile, the protonated polar groups form a dense hydrogen bond network with water molecules, maintaining the extended active conformation of the molecule—in particular, the rigid structure of the 5 thioether rings remains intact. These thioether rings are the core structural basis for Nisin to bind to bacterial cell membrane lipid Ⅱ and form pore channels.

Inhibition of Peptide Bond Hydrolysis Reduces Degradation RatePeptide bond hydrolysis is one of the main pathways for Nisin inactivation. Under strongly acidic conditions (pH < 3.0), the nucleophilic attack reaction of peptide bonds is significantly inhibited: the nucleophilicity of water molecules is reduced, making them unable to effectively attack the carbonyl carbon of peptide bonds; at the same time, the acidic environment can inhibit the activity of proteases (e.g., peptidases), reducing the risk of enzymatic degradation. In industry, Nisin is often dissolved in 0.02 mol/L hydrochloric acid for storage, which takes advantage of the stabilizing effect of strongly acidic conditions.

II. Weakly Acidic to Neutral Conditions (pH 4.0–7.0): Decreased Stability and Gradual Conformational Changes

As the pH increases to the weakly acidic to neutral range, the stability of Nisin decreases linearly, with its half-life shortened to several days to weeks at 25°C. The mechanism lies in conformational relaxation and partial degradation caused by charge imbalance:

Charge Neutralization Induces Molecular AggregationAt pH 4.0–7.0, the carboxyl groups in the molecule are gradually deprotonated (converted to -COO⁻), while the amino groups remain protonated, resulting in a reduction in the net positive charge of the molecule and a weakening of electrostatic repulsion. Hydrophobic amino acid residues (e.g., isoleucine, valine) are exposed, and molecules aggregate through hydrophobic interactions to form dimers or multimers. During aggregation, the spatial conformation of the Nisin molecule relaxes, and the relative positions of the thioether rings and peptide chains shift, leading to a decrease in its binding ability to bacterial lipid Ⅱ and a gradual attenuation of antibacterial activity.

Peptide Bond Hydrolysis Rate Rises SlowlyUnder neutral conditions, the nucleophilicity of water molecules increases, and peptide bond hydrolysis reactions begin to occur, with a higher probability of attacking the peptide bonds at the C-terminus of the molecule. Meanwhile, neutral pH is suitable for the activity of some microorganism-derived proteases, which may accelerate the enzymatic degradation of Nisin. For example, in a milk system at pH 7.0, the active half-life of Nisin is only 7–10 days, and further degradation needs to be delayed by refrigeration (4°C).

III. Weakly Alkaline to Strongly Alkaline Conditions (pH 8.0 and Above): Sharp Loss of Stability and Complete Structural Destruction

When pH > 8.0, the stability of Nisin drops sharply, and it can be completely inactivated within several hours. The core mechanisms are conformational collapse and large-scale hydrolysis of peptide bonds:

Charge Reversal Triggers Conformational DestructionUnder weakly alkaline to strongly alkaline conditions, amino acid residues are gradually deprotonated (-NH₂), and carboxyl groups are fully deprotonated (-COO⁻), giving the molecule an overall negative charge. The reversal of charge distribution disrupts the intramolecular salt bridge and hydrogen bond network, leading to irreversible stretching and distortion of the peptide chain. The rigid structure of the 5 thioether rings is destroyed—the thioether bonds are prone to oxidative cleavage under alkaline conditions. After the ring structure disintegrates, Nisin loses the spatial basis for binding to bacterial cell membranes, and its antibacterial activity disappears completely.

Rapid Peptide Bond Hydrolysis Causes Molecular FragmentationAlkaline conditions are the optimal environment for peptide bond hydrolysis: OH⁻ acts as a strong nucleophile, which can quickly attack the carbonyl carbon of peptide bonds and trigger peptide bond cleavage. Meanwhile, strong alkalinity (pH > 9.0) can directly induce autohydrolysis of the Nisin molecule, preferentially breaking the peptide bonds near the thioether rings and causing the molecule to split into inactive small peptide fragments. In addition, oxidation reactions in the alkaline environment will further damage amino acid residues (e.g., oxidation of methionine), exacerbating molecular inactivation.

IV. Synergistic Factors Affecting pH-Dependent Stability

Synergistic Effect of TemperatureElevated temperature intensifies the effect of pH on Nisin stability: under acidic conditions, high temperature (> 60°C) accelerates molecular motion, but due to stable conformation, the activity loss is relatively slow; while under alkaline conditions, high temperature sharply accelerates peptide bond hydrolysis and thioether ring cleavage. For example, at pH 9.0 and 80°C, Nisin can be completely inactivated within 10 minutes.

Regulatory Effect of Ionic StrengthHigh salt concentration (e.g., > 0.5 mol/L NaCl) compresses the electric double layer of Nisin molecules, exacerbating molecular aggregation under neutral conditions and reducing stability; whereas a low-salt environment can maintain the dispersed state of molecules and alleviate conformational relaxation under neutral pH.

Stabilizing Effect of Molecular ModificationIntroducing hydrophilic groups onto the Nisin molecular surface through chemical modification (e.g., PEGylation, phosphorylation) can enhance the anti-aggregation ability of the molecule under neutral conditions, while shielding peptide bonds from OH⁻ attack and broadening the stable pH range. For example, the stability of PEGylated Nisin at pH 6.0–7.0 is 3–5 times higher than that of unmodified Nisin.

The stability of Nisin follows the rule of stable in strong acid, attenuated in neutral, and inactivated in strong alkali with changes in pH. Its core mechanism is that pH affects the integrity of spatial conformation (especially the thioether ring structure) by regulating the molecular charge state, while altering the rates of peptide bond hydrolysis and oxidative degradation. In practical applications, strategies such as acidic pretreatment, low-temperature storage, or molecular modification should be selected according to the pH characteristics of the target system to maintain the structural and activity stability of Nisin.