Nisin is compounded with trehalose

As a natural food preservative, nisin is widely used in dairy and meat processing for its potent inhibitory effects against Gram-positive bacteria (e.g., Listeria monocytogenes, Staphylococcus aureus). However, its susceptibility to structural denaturation and inactivation during high-temperature processing (e.g., pasteurization, boiling) limits applications in heat-processed foods. Trehalose, a natural non-reducing disaccharide, exhibits "molecular chaperone" properties—it forms stable complexes with proteins/peptides to protect their spatial structure from heat-induced damage. When combined, trehalose significantly improves nisin’s thermal stability through three synergistic mechanisms: "intermolecular hydrogen bond anchoring," "hydration environment regulation," and "structural flexibility maintenance." This article analyzes the scientific principles underlying enhanced nisin thermal stability in the complex system from three perspectives—molecular interactions, thermal stability characterization, and mechanism validation—providing theoretical support for expanding nisin’s use in heat-processed foods.

I. Core Mechanism of Nisin Thermal Inactivation: Structural Denaturation and Active Site Destruction

To understand the protective effect of the complex system, it is first necessary to clarify the nature of nisin’s heat-induced inactivation: its antibacterial activity depends on a specific spatial structure (e.g., lanthionine rings), and high temperatures cause structural denaturation and active site damage, ultimately abolishing its bacteriostatic capacity.

(I) Heat-Induced Denaturation of Spatial Structure

The nisin molecule contains 5 lanthionine rings (cross-linked by special amino acids such as lanthionine and β-methyl lanthionine). These rings are critical for binding to bacterial cell membranes and forming pore channels. When temperatures exceed 60°C, non-covalent bonds (e.g., intramolecular hydrogen bonds, disulfide bonds) begin to break, causing lanthionine rings to unfold and the spatial structure to transition from a "compact globule" to a "loose linear chain":

Differential scanning calorimetry (DSC) shows pure nisin has a denaturation temperature (Tm) of ~72°C. Between 72–85°C, the proportion of secondary structures (α-helices, β-sheets) decreases from 35% to 12%, and the tertiary structure completely dissociates.

After heat-induced denaturation, nisin cannot bind to lipid II (a cell wall synthesis precursor) on bacterial membranes and loses pore-forming ability. Its antibacterial activity (minimum inhibitory concentration, MIC, against S. aureus) increases from 25 IU/mL to >100 IU/mL, losing practical value entirely.

(II) Chemical Damage to Active Sites

In addition to structural denaturation, high temperatures cause chemical modifications to nisin’s active sites (e.g., N-terminal lysine residues, C-terminal amino groups), further exacerbating inactivation:

At high temperatures, free amino groups in nisin easily undergo the Maillard reaction with carbonyl compounds (e.g., reducing sugars) in food systems, producing melanoidins that mask active sites.

Simultaneously, high temperatures accelerate nisin hydrolysis—peptide bond cleavage generates small molecular fragments lacking intact lanthionine rings, which cannot exert antibacterial effects.

II. Mechanisms of Enhanced Nisin Thermal Stability by Trehalose Complexation: Threefold Synergistic Protection

Trehalose’s thermal protection of nisin is not a single pathway but a threefold synergy of "intermolecular hydrogen bond anchoring," "hydration environment regulation to reduce denaturation," and "structural flexibility maintenance to resist heat"—inhibiting nisin’s thermal inactivation at the molecular level.

(I) Mechanism 1: Intermolecular Hydrogen Bond Anchoring Stabilizes Nisin’s Spatial Structure

Trehalose consists of two glucose units linked by an α,α-1,1-glycosidic bond. Multiple hydroxyl groups (-OH) on its surface form stable hydrogen bonds with nisin, acting as a "molecular scaffold" to prevent heat-induced structural unfolding:

Hydrogen bond binding sites: Acidic amino acid residues (e.g., aspartic acid, glutamic acid, containing -COOH) and hydroxyl-containing residues (e.g., threonine, serine) in nisin form hydrogen bonds (bond energy: ~20–30 kJ/mol) with trehalose’s hydroxyl groups. Molecular docking simulations show trehalose binds primarily around nisin’s lanthionine rings—particularly tightly to residues in rings 2 and 3—directly protecting key active structures.

Enhanced structural stability: In the complex system (nisin:trehalose = 1:5, mass ratio), nisin’s Tm increases from 72°C to 88°C. DSC curves show a 40% reduction in the endothermic peak area of denaturation, indicating significantly reduced heat-induced structural damage. Circular dichroism (CD) analysis reveals that after 30 minutes at 85°C, the secondary structure proportion of nisin in the complex group (28%) is much higher than in the pure nisin group (12%), with lanthionine ring integrity effectively maintained.

(II) Mechanism 2: Hydration Environment Regulation Reduces "Water-Induced" Thermal Denaturation

Water is a key medium for protein/peptide thermal denaturation—water molecules penetrate nisin’s interior, disrupting intramolecular hydrogen bonds and accelerating structural dissociation. Trehalose’s excellent "hydrating ability" regulates the hydration environment to reduce water-induced damage to nisin:

Replacing bound water: Trehalose’s hydroxyl groups form stronger hydrogen bonds with water molecules (1.5–2 times stronger than nisin-water bonds), preferentially binding to free and bound water in the system to form "trehalose-water" complexes. This reduces the number of water molecules around nisin, preventing penetration into its interior and structural disruption.

Reducing water activity (Aw): In the complex system, trehalose binding to water decreases the system’s Aw from 0.95 to 0.82 (at 1:5 mass ratio). In low-Aw environments, nisin’s thermal motion slows, and the rate of chemical changes (e.g., peptide bond hydrolysis, Maillard reaction) decreases by 30%–50%, further reducing active site damage.

(III) Mechanism 3: Maintaining Molecular Flexibility to Resist Heat-Induced Rigid Fracture

At high temperatures, pure nisin easily undergoes peptide bond fracture due to excessive rigidification. Trehalose maintains nisin’s molecular flexibility through interactions, enhancing heat resistance:

Flexibility regulation: After binding to trehalose, nisin’s molecular structure is moderately "relaxed" via hydrogen bonds, preventing excessive contraction and rigidity at high temperatures. Dynamic light scattering (DLS) shows that at 80°C, the particle size distribution of pure nisin shifts from 1.2–2.0 nm to 0.8–1.5 nm (rigid contraction), while the complex group retains a particle size of 1.1–1.9 nm, demonstrating significantly better molecular flexibility.

Reducing irreversible aggregation: Heat-denatured pure nisin easily forms irreversible aggregates (particle size >10 nm) with no antibacterial activity. Trehalose inhibits aggregation by maintaining nisin’s flexibility—after 30 minutes at 85°C, the complex group has only 15% of the aggregate content of the pure nisin group, with most nisin remaining in a monomeric state and retaining antibacterial activity.

III. Characterization and Validation of Thermal Stability in the Complex System: From In Vitro Tests to Application Scenarios

Enhanced thermal stability of the nisin-trehalose complex is confirmed via in vitro activity assays, structural characterization, and practical food application validation—providing data support for industrial use.

(I) In Vitro Thermal Stability Characterization: Significant Improvement in Activity Retention

Nisin’s antibacterial activity retention after heat treatment is used as a core indicator to compare thermal stability between pure nisin and the complex system:

Pasteurization (65°C, 30 min): Pure nisin retains 62% activity, while the complex system (1:5) retains 91%—its MIC only increases from 25 IU/mL to 30 IU/mL, maintaining effective bacteriostasis.

High-temperature boiling (100°C, 15 min): Pure nisin retains only 18% activity (essentially inactive), while the complex system retains 55%—its MIC of 50 IU/mL meets preservative requirements for moderately heat-processed foods.

Repeated heating (60°C, 30 min/day for 5 days): Pure nisin is completely inactivated by day 3, while the complex system retains 40% activity on day 5—showing advantages in foods requiring repeated heating (e.g., prepared meals).

(II) Molecular Structure Validation: Active Structure Retention After Heat Treatment

High-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyze the integrity of nisin’s molecular structure after heat treatment:

HPLC: After 30 minutes at 85°C, the main peak area of pure nisin decreases by 65% with multiple hydrolysis fragment peaks; the complex system’s main peak area decreases by only 20% with no obvious hydrolysis fragments, indicating trehalose inhibits nisin’s peptide bond cleavage.

MS: Nisin in the complex system retains a molecular weight of 3354 Da (natural nisin’s molecular weight), with no Maillard reaction-induced molecular weight shift (pure nisin’s molecular weight increases to >3500 Da due to the Maillard reaction)—confirming no chemical modification of active sites.

(III) Practical Application Validation: Preservative Efficacy in Heat-Processed Foods

Heat-processed meat products (e.g., sausages, processed at 90°C for 15 min) are used to validate the complex system’s practical efficacy:

Control group (no preservative): After 7 days of 4°C storage, total bacterial count (primarily S. aureus) reaches 10⁵ CFU/g with off-odors.

Pure nisin group (0.2 g/kg): Total bacterial count is 10⁴ CFU/g after 7 days, still posing a mild spoilage risk.

Complex group (0.2 g/kg nisin + 1.0 g/kg trehalose): Total bacterial count is only 10³ CFU/g after 7 days with no off-odors. Product texture and flavor remain unchanged—proving the complex system maintains nisin’s antibacterial activity in heat-processed foods without compromising quality.

IV. Optimization of Complex Ratio and Application Prospects

The nisin-trehalose ratio directly affects thermal stability enhancement and requires comprehensive optimization based on "activity retention," "cost control," and "food quality":

Optimal ratio: A nisin:trehalose mass ratio of 1:4–1:6 achieves the best thermal stability enhancement (>50% activity retention @100°C for 15 min). The trehalose dosage (0.8–1.2 g/kg) does not negatively affect food taste (e.g., sweetness) or texture.

Cost advantage: Trehalose is a natural food additive priced much lower than other chemical protectants (e.g., glycerol, sorbitol). The complex system costs only 15%–20% more than pure nisin but significantly extends the shelf life of heat-processed foods, offering higher overall cost-effectiveness.

With its enhanced thermal stability, the nisin-trehalose complex system effectively expands nisin’s application scope to heat-processed foods (e.g., baked goods, canned products, prepared meals), aligning with consumer demand for "natural, safe, and high-quality" food preservatives.