The REDOX properties of Nisin

As a natural antibacterial peptide produced by Streptococcus lactis, Nisin possesses unique redox properties conferred by specific functional groups (e.g., thioether bonds, amino groups, carboxyl groups) in its molecular structure. These properties are not only closely related to its own stability and antibacterial activity but also regulated by the external redox environment (e.g., oxygen concentration, pH value, metal ions). A detailed analysis can be conducted from four aspects: the molecular structural basis of redox properties, the characteristics of redox behavior, influencing factors, and the correlation with functions.

I. Molecular Structural Basis of Redox Properties

The core of nisin’s redox activity lies in the thioether bonds (lanthionine bonds, Lan; β-methyllanthionine bonds, MeLan) and terminal free groups (amino groups, carboxyl groups) in its molecule, among which the redox behavior of thioether bonds is the most critical feature:

The Nisin molecule consists of 34 amino acid residues and forms a compact cyclic structure through 5 thioether bonds. These thioether bonds are formed by the addition reaction between the sulfhydryl groups (-SH) of cysteine residues and the double bonds of dehydroalanine (Dha) or β-methyldehydroalanine (MeDha), essentially covalent bonds (-S-C-) formed between sulfur atoms and adjacent carbon atoms. The sulfur atoms in thioether bonds have moderate electronegativity, and their outer electron clouds are prone to changes in electron density (rather than typical "oxidation state increase/decrease") under the influence of an oxidizing environment, thereby participating in redox interactions. Meanwhile, the free amino group (-NH₂) at the N-terminus of the Nisin molecule can be oxidized to an imino group (-NH-) or further converted to a carbonyl group (C=O) under oxidizing conditions. Although the free carboxyl group (-COOH) at the C-terminus has weak redox activity, it can regulate the local redox microenvironment through proton transfer. In addition, the side chains of some amino acid residues in the molecule (e.g., the imidazole group of histidine) have a certain electron transfer capacity and can assist in the redox process.

II. Core Characteristics of Redox Behavior

Nisin’s redox properties do not manifest as a typical bidirectional reaction of "oxidizing agent/reducing agent" but are more inclined to "oxidative sensitivity"—that is, it is prone to structural modification in an oxidizing environment while maintaining or partially restoring its active structure in a reducing environment. The specific behaviors can be divided into two categories:

1. Structural Damage and Activity Decline Under Oxidizing Conditions

When exposed to oxidizing agents such as oxygen, hydrogen peroxide (H₂O₂), and sodium hypochlorite, the thioether bonds of nisin undergo preferential oxidative modification: the electron clouds of sulfur atoms are captured by oxidizing agents, leading to a decrease in the bond energy of thioether bonds and reduced structural stability. In severe cases, thioether bonds may break, causing the originally compact cyclic structure to depolymerize into linear fragments. This oxidative damage directly destroys the binding sites between nisin and bacterial cell membranes (especially lipid Ⅱ in Gram-positive bacteria), resulting in a significant decline in its antibacterial activity of inhibiting cell wall synthesis. For example, in a storage environment containing 5% oxygen, the antibacterial titer of Nisin decreases by 30%–40% within 28 days; if transition metal ions (which can catalyze oxidation reactions) such as Fe³⁺ and Cu²⁺ are present, the oxidation rate accelerates further, and the titer loss can exceed 50% within 7 days. In addition, the oxidation of the N-terminal amino group changes the molecular polarity, further weakening its hydrophobic interaction with cell membranes and exacerbating the loss of activity.

2. Structural Protection and Activity Maintenance Under Reducing Conditions

In an environment containing reducing substances (e.g., glutathione, vitamin C, sodium sulfite), the oxidative damage of Nisin can be significantly inhibited, and even some slightly oxidized molecules can recover their activity. Reducing substances can neutralize reactive oxygen species (ROS) in the environment by providing electrons, preventing their reaction with thioether bonds; at the same time, reducing agents can bind to the oxidized sulfur atoms in nisin molecules, repair the damaged electron cloud structure of thioether bonds, and maintain the integrity of the cyclic conformation. For example, adding 0.1% vitamin C as a reducing agent in food preservation can extend the shelf life of nisin by 2–3 times and reduce the antibacterial activity loss rate to below 10%; in vitro experiments have also confirmed that after nisin is slightly oxidized by low-concentration H₂O₂, its antibacterial activity can be restored to more than 85% of the initial level after adding 5 mmol/L glutathione.

III. Key Factors Influencing Nisin’s Redox Properties

Nisin’s redox behavior is not fixed but regulated by external environmental factors, among which pH value, metal ions, and oxygen concentration are the most important influencing factors:

1. Regulatory Role of pH Value

The pH value affects Nisin’s interaction with oxidizing/reducing agents by changing the charge state of its molecule. In an acidic environment (pH 2.0–5.0), the amino groups (-NH₂) in nisin molecules are protonated to form -NH₃⁺, while the carboxyl groups (-COOH) remain undissociated, making the molecule positively charged overall. At this time, the electron cloud density of thioether bonds is relatively high, and the resistance to oxidation is strong. In a neutral or alkaline environment (pH > 6.0), amino groups are deprotonated and carboxyl groups dissociate into -COO⁻, increasing molecular polarity. The electron clouds of thioether bonds are easily attacked by oxidizing agents, and the oxidation rate accelerates significantly. For example, the oxidation half-life of Nisin in a pH 7.0 buffer is only 1/3 of that at pH 3.0, which is the core reason for Nisin’s higher stability in acidic foods (e.g., yogurt, pickles).

2. Catalytic/Inhibitory Role of Metal Ions

Transition metal ions (e.g., Fe³⁺, Cu²⁺, Mn²⁺) can catalyze the generation of reactive oxygen species (e.g., ・OH) through the "Fenton reaction." These reactive oxygen species have much stronger oxidizing capacity than ordinary oxygen and accelerate the breakage of nisin’s thioether bonds. In contrast, certain metal ions (e.g., Zn²⁺, Ca²⁺) can bind to the carboxyl and amino groups in nisin molecules to form stable coordination compounds, enhancing the structural stability of thioether bonds and inhibiting oxidation reactions. For example, adding 0.02 mmol/L Zn²⁺ can reduce the activity loss rate of nisin in an oxidizing environment by 25%–30%, while the presence of Fe³⁺ can increase the oxidation rate by 1.5–2 times.

3. Synergistic Effect of Oxygen Concentration and Temperature

Oxygen is a direct inducer of nisin oxidation—the higher the concentration, the faster the oxidation reaction rate. In an oxygen-free environment (e.g., vacuum packaging), the titer of Nisin can remain stable for 6 months, while in an air environment, the titer can decrease by more than 20% within 1 month. Temperature regulates the activation energy of the oxidation reaction by affecting the rate of molecular movement: high temperatures (e.g., > 50℃) accelerate the collision frequency between nisin and oxygen and weaken the bond energy of thioether bonds, further exacerbating oxidative damage; low temperatures (e.g., 4℃ refrigeration) can significantly slow down the oxidation reaction and extend Nisin’s stable period.

IV. Correlation Between Redox Properties and Nisin’s Functions

Nisin’s redox properties are not an isolated chemical characteristic but directly affect its antibacterial function, application stability, and biological safety, specifically reflected in three aspects:

1. Decisive Role in Antibacterial Activity

Nisin’s antibacterial mechanism relies on its intact cyclic structure—only when thioether bonds are not oxidized and the conformation is stable can it bind to lipid Ⅱ on bacterial cell membranes, prevent cell wall synthesis, and form pores, leading to bacterial lysis. Once oxidative damage occurs, the cyclic structure is destroyed, the ability to bind to lipid Ⅱ is lost, and antibacterial activity disappears. Therefore, "maintaining redox stability" is a prerequisite for ensuring its antibacterial function.

2. Restrictions and Optimization for Application Scenarios

Based on the property that "Nisin has strong oxidation resistance in acidic environments and is prone to oxidation in alkaline/high-temperature environments," its applications are mainly concentrated in acidic foods (e.g., beverages, pickled products) or low-temperature processing scenarios. If it needs to be used in neutral foods (e.g., dairy products), it is necessary to combine it with reducing agents (e.g., vitamin C) or metal ion chelating agents (e.g., EDTA, which inhibits the catalytic effect of Fe³⁺ and Cu²⁺) to extend its stability by regulating the redox environment. This is also one of the core directions in the development of nisin application technologies.

3. Indirect Impact on Biological Safety

Nisin itself is a natural antibacterial peptide that can be degraded into amino acids by proteases in the human digestive tract, with extremely high safety. However, it should be noted that if nisin undergoes severe oxidation during storage or processing, a small amount of oxidative degradation products (e.g., short peptides containing oxidized thioether bonds) may be produced. Although there is currently no evidence that these products are toxic, from a safety perspective, reducing oxidative degradation by controlling the redox environment (e.g., low temperature, light protection, adding reducing agents) remains an important measure to ensure the safety of nisin applications.

The redox properties of nisin are centered on the "oxidative sensitivity of thioether bonds," manifested as structural damage and activity decline in an oxidizing environment, and structural protection and activity maintenance in a reducing environment. These properties are significantly regulated by factors such as pH value, metal ions, and oxygen concentration. They not only determine nisin’s antibacterial activity and application stability but also provide a key theoretical basis for optimizing application technologies (e.g., formulation design, stabilizer selection) in fields such as food preservation and biomedicine. In the future, in-depth research on the molecular mechanism of nisin’s redox reactions is expected to lead to the development of more efficient oxidation protection strategies, further expanding its application scope.