The stability of Nisin in food processing

As a natural antibacterial peptide, nisin is widely used in food processing to inhibit the growth of Gram-positive bacteria (such as Listeria and Staphylococcus) and extend food shelf life. However, its stability is susceptible to processing technologies, food system environments, and storage conditions, which may lead to reduced activity or inactivation. Therefore, targeted control strategies are required to ensure its application effectiveness, with specific measures analyzed from the following aspects:

I. Regulation of the Basic Environment of Food Systems

The pH value, water activity (aₙᵥ), and component composition of food itself are core fundamental factors affecting Nisin stability. Priority should be given to reducing activity loss through environmental regulation.

1. pH Value Regulation

Nisin exhibits significantly higher stability in acidic environments, which is closely related to its molecular structural characteristics:

In acidic conditions, basic amino acid residues (such as lysine and histidine) in nisin molecules are less prone to deprotonation, maintaining a stable peptide chain conformation and resisting degradation or polymerization.

In neutral or alkaline environments (pH > 7.0), peptide bonds are easily activated by hydrolases, and the molecular conformation stretches due to changes in charge distribution, leading to a rapid decline in antibacterial activity.

Thus, nisin can naturally maintain high stability in the processing of acidic foods (e.g., yogurt, pickled vegetables, acidic beverages). For neutral/alkaline foods (e.g., fresh-cut vegetables, some meat products), food-grade acids (such as citric acid and lactic acid) should be added to adjust the system pH to 3.0–6.0. Alternatively, Nisin can be added during acidic pre-processing steps (e.g., marination, seasoning) to avoid prolonged exposure to high-pH environments.

2. Water Activity (aₙᵥ) Control

The impact of aₙᵥ on nisin stability is most prominent during storage:

High aₙᵥ (> 0.6) provides conditions for microbial growth, enzymatic reactions (e.g., protease hydrolysis), and chemical degradation (e.g., oxidation, peptide bond breakage), accelerating Nisin inactivation.

Low aₙᵥ (< 0.3) slows molecular movement and significantly inhibits the above reactions.

For dry foods (e.g., baked goods, dehydrated meat products), drying technologies (e.g., hot-air drying, vacuum drying) should be used to control aₙᵥ at 0.2–0.5, while preventing moisture absorption after processing. For liquid foods (e.g., liquid milk, beverages), low-temperature storage (0–4℃) should be combined to reduce moisture-mediated degradation.

3. Mitigation of Component Interactions

Food components may interact with Nisin:

Fat molecules may encapsulate nisin, reducing its migration efficiency to microbial cells.

Metal ions (e.g., Ca²⁺, Mg²⁺) may bind to carboxyl and amino groups in nisin, altering its conformational stability.

Macromolecules (e.g., proteins, polysaccharides) may non-specifically adsorb nisin, reducing the amount of free active forms.

To address this, the order of component addition can be adjusted (e.g., adding Nisin first to ensure uniform dispersion before adding fats or macromolecular excipients), or the content of metal ions can be controlled (e.g., avoiding excessive use of calcium phosphate as a stabilizer) to minimize stability loss caused by interactions.

II. Optimization of Processing Parameters

Processing technologies such as heat treatment, high-pressure processing, and irradiation may damage Nisin’s molecular structure or accelerate its degradation. Parameter optimization is needed to balance "sterilization efficiency" and "Nisin activity retention."

1. Heat Treatment Optimization

Heat treatment is one of the most common causes of nisin inactivation:

High temperatures (> 80℃) induce thermal denaturation of nisin’s peptide chain, destroying its unique "lanthionine ring structure" (critical for binding to bacterial cell membranes and exerting antibacterial effects). Longer heating time and higher temperature lead to more significant activity loss (e.g., 121℃ high-pressure sterilization for 30 minutes can reduce nisin activity by over 80%).

Thus, heat treatment conditions should be adjusted based on food type:

For foods requiring high-temperature sterilization (e.g., canned foods), "low-temperature long-time" or "ultra-high-temperature instantaneous sterilization (UHT, 135–150℃, 2–5 seconds)" can be used. Although UHT uses high temperatures, its extremely short duration reduces nisin’s heat exposure, retaining 30%–50% more activity compared to traditional sterilization.

For non-sterile foods (e.g., low-temperature meat products), heat treatment temperature can be controlled at 60–70℃, or Nisin can be added after heat treatment during the cooling phase (< 40℃) to completely avoid thermal damage.

2. High-Pressure Processing (HHP) Adjustment

HHP (typically 200–600 MPa) is a non-thermal processing technology. While it does not directly break Nisin’s peptide bonds, high pressure may cause reversible or irreversible changes in nisin’s conformation:

Pressure < 400 MPa: Most conformational changes are reversible, and activity can be restored after pressure release.

Pressure > 500 MPa: Irreversible changes in peptide chain folding may occur, destroying antibacterial active sites.

When combining HHP with nisin, pressure should be controlled at 300–400 MPa, holding time shortened (< 10 minutes), and Nisin added after HHP treatment to avoid conformational damage from high pressure.

3. Irradiation Dosage Control

Irradiation (e.g., γ-rays, electron beams) is mainly used for food sterilization and preservation:

Low-dose irradiation (< 5 kGy) has little impact on Nisin stability and may even indirectly extend its effective duration by reducing microbial consumption of Nisin.

High-dose irradiation (> 10 kGy) generates free radicals (e.g., hydroxyl radicals, superoxide anions) that attack amino acid residues (e.g., methionine, tyrosine) in Nisin, causing peptide bond breakage or oxidative degradation.

Thus, when applying nisin in irradiated foods, irradiation dosage should be controlled below 5 kGy, or free radical scavengers (e.g., vitamin C, tea polyphenols) added to reduce oxidative damage.

III. Application of Formulation Modification and Encapsulation Technology

Constructing a "protective barrier" around nisin molecules through formulation modification or encapsulation technology reduces contact with adverse external environments (e.g., high temperature, high pH, enzymes), serving as an efficient means to enhance stability.

1. Formulation Modification: Microcapsules and Nanoparticles

Preparing nisin into microcapsules or nanoparticles is a mainstream approach, with common encapsulation materials including:

Natural polysaccharides (e.g., chitosan, sodium alginate): These materials have good pH sensitivity—stable in acidic environments and capable of slow nisin release in neutral/alkaline food systems, while preventing degradation by alkaline conditions.

Proteins (e.g., whey protein, gelatin): Thermal denaturation of proteins forms dense microspheres, encapsulating Nisin to resist high temperatures (< 80℃) and protease attacks.

Lipids (e.g., soybean oil, monoglycerides): Lipid-based materials (e.g., liposomes) simulate biomembrane structures, encapsulating Nisin in lipid bilayers to reduce interactions with food fats and metal ions.

For example, nisin microcapsules prepared via sodium alginate-chitosan complex coacervation retain over 60% more activity than free Nisin after 14 days of storage in pH 8.0 meat products. Nisin nanoparticles encapsulated in whey protein lose only 1/3 of the activity of free nisin after heating at 90℃ for 10 minutes.

2. Compound Formulation with Natural Ingredients

Compounding nisin with other natural ingredients (e.g., plant extracts, probiotics) can enhance stability through synergistic effects:

Compounding with rosemary extract: Phenolic compounds in rosemary scavenge free radicals in food systems, reducing oxidative degradation of Nisin.

Compounding with lactic acid bacteria: Organic acids produced by lactic acid bacteria maintain an acidic system environment, indirectly stabilizing Nisin’s conformation.

Such compounding not only improves stability but also broadens the antibacterial spectrum (e.g., inhibiting some Gram-negative bacteria), further enhancing food preservation effects.

IV. Strict Control of Storage Conditions

Even if nisin stability is ensured during processing, improper storage conditions may still cause activity loss. Control should focus on three core factors: temperature, light, and oxygen.

1. Temperature Control

Temperature is critical for Nisin stability during storage:

Room temperature (25–30℃): Fast molecular movement of nisin leads to slow degradation, with 20%–30% activity loss possible after 1 month of storage.

Low temperature (0–4℃): Significantly slows molecular movement and degradation reactions, controlling activity loss within 10% after 3 months of storage.

Freezing (-18℃ or below): Almost no activity loss occurs, allowing long-term storage.

Thus, Nisin-containing foods (especially liquid or high-moisture foods) should be prioritized for low-temperature or frozen storage, avoiding prolonged room-temperature placement. For dry foods (e.g., nisin premixes), while storage at room temperature (< 25℃) is feasible, repeated freeze-thaw cycles should be avoided (ice crystals formed during freeze-thaw may damage nisin’s conformation).

2. Light Protection

Light (especially ultraviolet and visible light) damages Nisin molecules through photooxidation:

Ultraviolet light directly excites amino acid residues (e.g., tryptophan, tyrosine) in Nisin, causing peptide bond breakage.

Visible light may generate singlet oxygen, triggering oxidative degradation of Nisin.

Thus, nisin-containing foods should use light-proof packaging materials (e.g., brown glass bottles, opaque plastic films) and be stored in cool, dark places (e.g., inside warehouse shelves, avoiding direct sunlight).

3. Oxygen Isolation

Oxygen accelerates the oxidative degradation of nisin, and this effect is exacerbated under high temperatures and light:

For oxygen-sensitive foods (e.g., high-fat meat products, nuts), vacuum packaging or nitrogen-filled packaging should be used to reduce oxygen content in packages (controlling oxygen below 1%).

For nisin premixes (e.g., powdered products), sealed packaging should be used, with desiccants (e.g., silica gel) and oxygen absorbers (e.g., iron powder oxygen absorbers) placed inside. Package damage should also be avoided to prevent oxygen ingress.