The mechanism of action of Nisin with food components

As a widely used natural food preservative, nisin owes its stability, antibacterial activity, and final efficacy in food systems not only to its intrinsic properties but also to its complex interactions with various food components (proteins, lipids, carbohydrates, minerals, food additives, etc.). These interactions directly affect nisin’s antibacterial performance by altering its molecular conformation, solubility, aggregation state, or the exposure of active sites. The core mechanisms can be analyzed by categorizing food components as follows:

I. Interactions with Protein Components: Active Site Shielding or Conformational Changes

Proteins in food (e.g., milk proteins, meat proteins, plant proteins) are among the most frequent interactants with nisin. Their interactions, primarily non-covalent binding, often lead to reduced antibacterial activity of nisin, with key mechanisms including:

1. Electrostatic Binding and Active Site Shielding

In the acidic or neutral environments common in food, nisin molecules carry a net positive charge (containing 2 lysine residues, with an isoelectric point of ~8.0). Most food proteins (e.g., whey protein, soy protein) carry a negative charge in this pH range due to the dissociation of carboxyl groups (-COOH). The two can form stable complexes through electrostatic interactions. This binding shields nisin’s key active sites (e.g., hydrophobic segments that bind to bacterial lipid Ⅱ, and domains involved in transmembrane pore formation), preventing nisin from binding to the cell membranes of target bacteria and significantly reducing its antibacterial activity. For example, in dairy products, the negatively charged regions of casein can tightly bind to the positively charged lysine residues of nisin, lowering the concentration of free nisin. Thus, higher Nisin addition is required to maintain antibacterial efficacy.

2. Hydrophobic Interactions and Molecular Aggregation

Protein molecules (especially denatured proteins, such as heated milk proteins) expose a large number of hydrophobic amino acid residues. Nisin molecules themselves also contain hydrophobic segments (e.g., regions enriched with alanine and valine). The two can further promote complex formation through hydrophobic interactions, and even trigger Nisin-protein co-aggregation. Aggregated nisin molecules have limited spatial mobility, making it difficult for them to diffuse to bacterial surfaces. Additionally, active sites are encapsulated within aggregates, preventing specific binding to lipid Ⅱ, ultimately "inactivating" Nisin’s antibacterial activity.

3. Hydrogen Bond-Mediated Conformational Changes

Polar groups (e.g., hydroxyl, amino, carbonyl groups) of some proteins can form hydrogen bonds with the peptide bond carbonyl groups and the hydroxyl groups of serine/threonine in nisin molecules. This binding may alter nisin’s spatial conformation—Nisin’s antibacterial activity relies on its rigid "hairpin-like" structure formed by lanthionine cross-links. Hydrogen bond formation can disrupt this conformation, reducing the specificity of Nisin’s binding to lipid Ⅱ and thereby weakening its antibacterial ability.

II. Interactions with Lipid Components: Competitive Target Binding or Altered Dispersibility

Interactions between lipids in food (e.g., vegetable oil, animal fat, milk fat) and Nisin are "bidirectional": they may reduce antibacterial activity through competitive binding or improve stability by enhancing dispersibility, depending on the type and content of lipids:

1. Non-Specific Binding with Free Fatty Acids

The hydrophobic chains of free fatty acids in food (e.g., oleic acid, palmitic acid produced by vegetable oil hydrolysis) can bind to nisin’s hydrophobic segments via hydrophobic interactions. This binding mechanism is similar to Nisin’s binding to bacterial lipid Ⅱ (both rely on hydrophobic interactions), meaning free fatty acids "compete" to occupy Nisin’s hydrophobic active sites. As a result, nisin cannot bind to lipid Ⅱ on bacterial cell membranes, and its antibacterial activity is competitively inhibited. For example, in high-fat meat products, the presence of large amounts of free fatty acids reduces the effective concentration of nisin. Countermeasures include adjusting formulations (e.g., reducing fat content) or using microencapsulation technology to minimize such interactions.

2. Adsorption and Dispersion with Lipid Bilayers

If lipids in food exist in an emulsified state (e.g., lipid bilayer structures in emulsions), nisin molecules can adsorb to the surface of lipid bilayers via hydrophobic interactions:

On one hand, this adsorption reduces nisin’s binding to proteins, increasing the proportion of free Nisin.

On the other hand, lipid bilayers can act as "carriers" for Nisin, helping it disperse uniformly in the food system and avoiding activity loss caused by self-aggregation.

For example, in plant protein beverages, emulsified soybean oil particles can adsorb Nisin, enabling it to diffuse more easily throughout the system and enhancing the inhibition of contaminating bacteria. However, it should be noted that excessive lipid content may lead to over-adsorption of nisin on lipid surfaces, preventing it from contacting bacteria and instead reducing activity.

III. Interactions with Carbohydrate Components: Stability Regulation and Diffusion Barriers

Carbohydrates (e.g., starch, sucrose, pectin, dietary fiber) are major fillers and thickeners in food. Their interactions with nisin are primarily physical binding or environmental regulation, and their impact on nisin’s activity is mostly indirect:

1. Hydrogen Bond Binding and Stability Enhancement

Polysaccharide molecules such as starch and pectin contain a large number of hydroxyl groups (-OH), which can form hydrogen bonds with the peptide bond carbonyl groups and polar amino acid side chains of Nisin. While this binding does not directly shield Nisin’s active sites, it enhances nisin’s molecular stability—hydrogen bonds reduce conformational damage during processing (e.g., heating, stirring) and lower the probability of Nisin degradation by proteases (the coating formed by polysaccharides blocks proteases from contacting Nisin). For example, in baked goods, hydrogen bond binding between starch and Nisin allows nisin to retain some activity after high-temperature baking, extending the food’s shelf life.

2. High-Viscosity Environment and Diffusion Barriers

When carbohydrate content in food is high (e.g., high-sugar jams, high-starch pastes), the system viscosity increases significantly, which physically hinders Nisin’s diffusion. Nisin must diffuse to bacterial surfaces to exert its effect, but a high-viscosity environment slows down its molecular movement. This prevents Nisin from timely contacting contaminating bacteria in the system, especially in the interior or local regions of food, potentially forming "antibacterial blind spots" and weakening overall preservative efficacy. Additionally, insoluble carbohydrates such as dietary fiber may immobilize Nisin through adsorption, further limiting its diffusion range.

IV. Interactions with Minerals and Metal Ions: Charge Neutralization or Conformational Stabilization

Interactions between minerals in food (e.g., calcium, magnesium, iron, zinc ions, mostly from raw materials or food fortifiers) and Nisin primarily occur via electrostatic interactions, affecting Nisin’s solubility and activity:

1. Charge Neutralization by Anionic Metal Complexes

If food contains anions such as phosphates or sulfates (often bound to calcium or magnesium ions), or directly added high-valent metal ions (e.g., Fe³⁺, Zn²⁺ in fortified foods), electrostatic interactions with Nisin may occur. Nisin carries a positive charge, so anions or negatively charged metal complexes (e.g., Fe(OH)₃ colloids) can reduce Nisin’s surface positive charge through charge neutralization. This weakens Nisin’s electrostatic attraction to bacterial cell membranes (which are negatively charged) and may promote Nisin molecular aggregation (due to reduced charge repulsion), leading to decreased activity. For example, in iron-fortified milk, the binding of Fe³⁺ to Nisin reduces Nisin’s antibacterial activity by approximately 20%–30%.

2. Conformational Stabilization by Divalent Cations

Low concentrations of divalent cations such as calcium and magnesium (e.g., Ca²⁺ in dairy products) have a protective effect on Nisin’s activity: these ions can form coordinate bonds with the carboxyl groups (aspartic acid residues) of Nisin molecules and stabilize Nisin’s "hairpin-like" conformation via electrostatic interactions, reducing insufficient exposure of active sites caused by conformational relaxation. Studies have shown that in food systems containing 0.01–0.05 mol/L Ca²⁺, Nisin’s inhibitory effect on Bacillus cereus is approximately 15% stronger than in systems without Ca²⁺. However, at excessively high concentrations (e.g.,>0.1 mol/L), Ca²⁺ binds to Nisin excessively, 反而 reducing its activity.

V. Interactions with Food Additives: Synergistic Enhancement or Antagonistic Inhibition

Interactions between common food additives (e.g., organic acids, EDTA, other preservatives) and Nisin are complex—some can achieve synergistic enhancement, while others may cause antagonistic inhibition:

1. Synergistic Enhancement by Organic Acids

Organic acids such as citric acid, lactic acid, and acetic acid are commonly used acidulants in food. Their synergistic mechanism with Nisin mainly includes two aspects:

First, organic acids lower the food’s pH, enhancing the positive charge of Nisin molecules (moving away from the isoelectric point), improving solubility and the proportion of free Nisin, while strengthening Nisin’s electrostatic attraction to bacterial cell membranes.

Second, organic acids disrupt the integrity of bacterial cell membranes (e.g., altering lipid bilayer fluidity), making it easier for Nisin to insert into the membrane and form transmembrane pores. The combination of the two significantly enhances antibacterial efficacy. For example, in pickled vegetables, the combined use of lactic acid and Nisin can reduce the inhibitory concentration against Listeria by more than 50%.

2. Auxiliary Effects by Chelating Agents (e.g., EDTA)

EDTA (ethylenediaminetetraacetic acid) is a commonly used metal chelating agent. It has a certain inhibitory effect on Gram-negative bacteria by itself, and when used in combination with Nisin, it can chelate divalent cations (e.g., Ca²⁺, Mg²⁺) on bacterial cell membranes, disrupting membrane stability and facilitating Nisin penetration. Meanwhile, EDTA can chelate free metal ions in food, reducing their antagonistic interactions with Nisin and indirectly enhancing Nisin’s activity.

3. Antagonistic Inhibition by Reductive Additives

Reductive additives such as sulfites and vitamin C (ascorbic acid) may undergo redox reactions with Nisin: the disulfide bonds (lanthionine cross-links) in Nisin molecules are critical for maintaining its conformation and activity. Reductive substances can break these disulfide bonds, leading to Nisin’s conformational disintegration and activity loss. Therefore, in foods containing sulfites (e.g., certain preserved fruits, wine), Nisin’s antibacterial effect is significantly weakened, and their simultaneous use should be avoided.

The interaction mechanisms between Nisin and food components revolve around "molecular binding – conformational change – activity regulation":

Proteins shield active sites via electrostatic and hydrophobic interactions;

Lipids affect activity through competitive binding or dispersion regulation;

Carbohydrates indirectly alter stability and diffusivity via hydrogen bonds and viscosity;

Minerals regulate solubility via charge effects;

Food additives may either synergistically enhance or antagonistically inhibit Nisin’s activity.

These interactions indicate that in practical applications, Nisin’s addition amount and usage method (e.g., microencapsulation, combined use with organic acids) should be optimized based on the food formula (e.g., protein/fat content, pH, additive type) to maximize its antibacterial efficacy while ensuring food safety and quality.