Optimize the stability of Nisin

As a natural antibacterial peptide, the stability of nisin directly determines its application efficacy in fields such as food processing and biological preservation. Temperature, pH, and metal ions are the core environmental factors affecting its stability; by regulating nisin’s molecular structure (e.g., secondary structure integrity, charge state) and aggregation behavior, these three factors collectively determine its activity retention rate. In-depth analysis of the mechanisms of these factors and targeted optimization are key to achieving the efficient application of nisin, with specific research as follows:

I. Impact of Temperature on nisin Stability and Optimization Strategies

Temperature affects the structural stability of nisin by accelerating molecular thermal motion and breaking chemical bonds (e.g., hydrogen bonds, thioether bonds). Its effect is closely related to heating temperature, duration, and synergistic environments (e.g., pH, water activity).

When no other conditions are regulated, nisin’s thermal stability exhibits an obvious "temperature threshold effect":

When the temperature is below 60°C, molecular motion is slow, and the structure is less prone to denaturation. Even after long-term treatment (e.g., 24 hours), the activity retention rate can still reach over 85%.

When the temperature rises to 80–100°C (conventional thermal processing temperature), if in a neutral or alkaline environment, the internal cyclic structure of nisin molecules (maintained by thioether bonds) is prone to breaking. Hydrophobic regions are exposed and cause aggregation, leading to more than 40% activity loss within 10 minutes.

Under 121°C high-pressure sterilization conditions, without protective measures, Nisin activity can drop to less than 20% of the initial value within 20 minutes, basically losing its antibacterial ability.

Optimization strategies for temperature should focus on "reducing the rate of thermal denaturation":

Combined with food pH regulation: Maintain the system pH in the acidic range of 2.0–5.0. At this point, nisin molecules are positively charged, the stability of hydrogen bonds and thioether bonds is enhanced, and the thermal denaturation temperature is significantly increased. For example, in fruit juice at pH 3.0, after 20 minutes of sterilization at 121°C, the activity retention rate can increase from 30% (under neutral conditions) to over 70%.

Control water activity: Process Nisin into powder formulations or apply it to low-water-activity foods (e.g., dehydrated vegetables). This slows molecular motion, reduces heat sensitivity, and allows stable storage at room temperature for 1–2 years. Even after heating at 80°C for 10 minutes, activity loss can be controlled within 20%.

Add protective agents: Incorporate 0.5%–1% sucrose, maltodextrin, or casein into liquid foods. These substances can form hydrogen bonds with nisin, encapsulate the molecules, and reduce heat-induced aggregation. Under heating at 100°C, the activity retention rate can be increased by 30%–40%.

II. Impact of pH on Nisin Stability and Optimization Strategies

pH is a core factor regulating nisin’s molecular charge state and structural integrity. Its impact on stability runs through the entire process of storage and processing, and it is synergistically associated with solubility and antibacterial activity.

Acidic range (pH 2.0–5.0): The amino groups (-NH₂) in nisin molecules are easily protonated to form -NH₃⁺, resulting in an overall positive charge. This charged state not only enhances interaction with polar solvents (improving solubility) but also reduces intermolecular hydrophobic aggregation while maintaining the stability of the cyclic structure. Within this pH range, even when stored at 4°C (refrigeration) or 25°C (room temperature) for 1 month, nisin activity loss is only 5%–10%, and it can still maintain high antibacterial activity after low-to-medium temperature processing.

Neutral range (pH 6.0–7.0): The degree of molecular protonation decreases, positive charge reduces, hydrophobic regions are gradually exposed, and slight aggregation begins to occur. The rate of activity degradation accelerates during storage—activity loss can reach 20%–30% after 1 month of storage at 25°C, and thermal stability also decreases accordingly.

Alkaline range (pH > 8.0): Molecules are completely deprotonated, positive charge disappears, and hydrophobic interactions dominate molecular aggregation, forming insoluble precipitates. At the same time, the cyclic structure breaks due to reduced bond energy caused by deprotonation, leading to structural depolymerization. Even under refrigeration at 4°C, more than 50% of the activity can be lost within 1 week, basically failing to meet application requirements.

Optimization strategies for pH should focus on "maintaining an acidic microenvironment" or "inhibiting structural degradation under alkaline conditions":

For inherently acidic foods (e.g., yogurt, pickles, acidic beverages): The natural pH environment (usually pH 3.5–4.5) can be directly utilized without additional regulation. Only the introduction of alkaline excipients (e.g., sodium bicarbonate) during processing needs to be avoided.

For neutral or weakly alkaline foods (e.g., milk, soy products, meat products):

Adjust the system pH to below 5.0 by adding food-grade organic acids (e.g., citric acid, lactic acid).

Adopt "microencapsulation technology": Encapsulate nisin in pH-sensitive wall materials (e.g., sodium alginate, chitosan). The wall materials remain stable in neutral/alkaline environments to prevent Nisin from contacting external alkaline substances, and release Nisin only when entering the target environment (e.g., human intestines or acidic food simulation systems). This not only protects nisin’s structural stability but also does not affect the food’s pH and flavor.

For alkaline food processing: Add small amounts of acidic antioxidants (e.g., ascorbic acid) to inhibit the destruction of nisin structure by oxidative stress and assist in improving stability. For example, adding 0.1% ascorbic acid to milk at pH 7.0 can reduce the activity loss from 30% to approximately 15% after 1 month of storage at 25°C.

III. Impact of Metal Ions on Nisin Stability and Optimization Strategies

Metal ions affect nisin’s structural stability by forming coordination bonds with groups such as carboxyl and amino groups in Nisin molecules or changing the system’s ionic strength. Different types of metal ions have significantly different effects, which can be divided into two categories: "stabilization-promoting" and "degradation-promoting."

1. Stabilization-Promoting Metal Ions

These mainly include monovalent cations (e.g., Na⁺, K⁺) and some divalent cations (e.g., Mg²⁺):

Low concentrations (0.05–0.1 mol/L) of Na⁺ or K⁺ can enhance the interaction between nisin and water molecules through the "salting-in effect," reducing molecular aggregation. Meanwhile, their positive charge can form weak electrostatic interactions with the negative charge on the nisin molecular surface (from partial deprotonation of carboxyl groups), stabilizing the molecular conformation. For example, in a pH 4.0 solution containing 0.05 mol/L NaCl, the activity retention rate of nisin after 1 month of storage at 25°C is 10%–15% higher than that of the salt-free group, and the activity loss after heating at 80°C for 10 minutes is also reduced by 8%–10%.

Mg²⁺ can further strengthen the cyclic structure by forming coordination bonds with carboxyl groups in nisin molecules. When 0.02 mol/L Mg²⁺ is present, even if the pH rises to 6.0, the thermal stability of nisin can still be close to that of the magnesium-free group at pH 5.0.

2. Degradation-Promoting Metal Ions

These are mainly divalent or trivalent heavy metal ions (e.g., Cu²⁺, Fe³⁺, Zn²⁺), with mechanisms including:

At high concentrations (>0.01 mol/L), the strong coordination ability of heavy metal ions damages the thioether bonds and hydrogen bonds in nisin molecules, leading to cyclic structure breakage and molecular depolymerization. For example, in a solution containing 0.02 mol/L Cu²⁺, the activity loss of nisin after 1 week of storage at 4°C can reach over 40%, much higher than 5% in the copper-free group.

Some metal ions (e.g., Fe³⁺) can catalyze oxidation reactions in the system, generating free radicals that further oxidize nisin’s amino acid residues (e.g., methionine) and accelerate activity degradation.

In addition, the effect of Ca²⁺ is "concentration-dependent": low concentrations (<0.01 mol/L) can slightly promote stability, while high concentrations (>0.05 mol/L) can reduce stability by competing with nisin for water molecules or causing aggregation.

3. Optimization Strategies for Metal Ions

Differentiated designs based on ion types are required:

Directionally introduce stabilization-promoting ions: Add an appropriate amount (0.05–0.1 mol/L) of NaCl, KCl, or MgCl₂ to nisin formulations or food systems. Especially in neutral foods, this can help offset the adverse effects of increased pH on stability.

Inhibit the effect of degradation-promoting ions: For foods that may introduce heavy metal ions (e.g., meat products, seafood products), add metal chelating agents (e.g., EDTA, citric acid) in advance. These agents form stable chelates with Cu²⁺, Fe³⁺, etc., preventing their interaction with Nisin. Adding 0.01 mol/L EDTA can increase the storage stability of Nisin in Cu²⁺-containing systems by more than 30%.

Control ion concentration: Avoid excessively high total concentrations of metal ions in the system (especially Ca²⁺). Optimize food formulations (e.g., reduce the amount of calcium salt additives) or use ion exchange technology to lower the system’s ionic strength, ensuring the structural stability of nisin.

IV. Core Logic of Multi-Factor Synergistic Optimization

The stability of nisin is not affected by a single factor but by the synergistic effect of temperature, pH, and metal ions. For example, under alkaline conditions, the degradation effect of heavy metal ions on nisin is significantly enhanced, while an acidic environment can partially offset the negative effects of high temperature and heavy metals. Therefore, in practical optimization, a "multi-factor synergistic regulation" mindset must be established:

Prioritize pH adjustment (maintaining 2.0–5.0) to lay a stable foundation.

Combine with processing temperature control (mainly low-to-medium temperatures, with protective agents added if necessary) and metal ion management (introducing stabilization-promoting ions, chelating harmful ions).

Form a combined strategy of "pH regulation + temperature protection + ion optimization."

For example, when applying nisin in neutral milk: first, add lactic acid to adjust the pH to 5.0; then add 0.05 mol/L NaCl and 0.01 mol/L EDTA; finally, use pasteurization (60–65°C, 30 minutes) instead of high-temperature sterilization. In this case, the activity retention rate of nisin can increase from 50% (with single pH regulation) to over 80%, while balancing the food’s flavor and safety.

The stability optimization of nisin requires an in-depth understanding of the mechanisms of temperature, pH, and metal ions. By targeted regulation of environmental parameters, introduction of protective agents, or adoption of formulation technologies, it is possible to meet food processing needs while maximizing the retention of nisin’s antibacterial activity, providing support for the industrial application of natural preservation technology.