The preservation effect of the combination of Nisin and lysozyme

Chilled meat products (e.g., chilled ham, sausages, soy-braised beef) retain nutrients and flavor well due to their low processing temperature (typically 60–85°C). However, this also results in incomplete sterilization, making them vulnerable to microbial contamination and spoilage. Among the contaminants, Gram-positive bacteria (e.g., Listeria monocytogenes, Staphylococcus aureus) and Gram-negative bacteria (e.g., E. coli, Pseudomonas) are the main spoilage and pathogenic microorganisms. Under room temperature, the shelf life of chilled meat products is only 1–2 days, and even under 4°C refrigeration, it often lasts less than 15 days.

Nisin (a natural antimicrobial peptide) exhibits significant inhibitory effects against Gram-positive bacteria, while lysozyme (a broad-spectrum antimicrobial enzyme) destroys bacterial cell walls and acts on both Gram-positive and some Gram-negative bacteria. Recent studies have shown that the combination of Nisin and lysozyme enhances the preservation effect of chilled meat products through "synergistic antimicrobial activity," extending shelf life and ensuring safety. This article systematically analyzes the preservation value of the Nisin-lysozyme combination in chilled meat products from three aspects—synergistic antimicrobial mechanisms, efficacy evaluation dimensions, and practical application optimization—providing references for industrial applications.

I. Synergistic Antimicrobial Mechanisms of the Nisin-Lysozyme Combination: Targeted Breakthrough of Microbial Resistance Barriers

Spoilage microorganisms in chilled meat products are mainly bacteria, and their resistance primarily stems from cell wall structures and enzymatic defense systems. The combination of Nisin and lysozyme is not a simple superposition of effects but a "divided and collaborative" disruption of bacterial defenses, with specific mechanisms divided into three steps:

(I) Lysozyme: "First Step of Cell Wall Breakdown" to Disintegrate Structures and Open Antimicrobial Channels

The core target of lysozyme is the peptidoglycan in bacterial cell walls (the main component of Gram-positive bacterial cell walls and also present in the inner layer of Gram-negative bacterial cell walls):

Lysozyme specifically hydrolyzes the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, causing the peptidoglycan chain to break. For Gram-positive bacteria (e.g., Listeria monocytogenes), whose cell walls lack an outer membrane for protection, peptidoglycan hydrolysis directly leads to cell wall collapse and "protoplast formation" (cells lose structural support and easily rupture due to osmotic imbalance).

For Gram-negative bacteria (e.g., E. coli), lysozyme cannot directly penetrate the outer membrane, but it can hydrolyze the connection sites between outer membrane proteins and peptidoglycan, disrupting the binding stability of the outer membrane and cell wall and creating tiny pores in the outer membrane. Experiments show that after treating E. coli with 0.1% lysozyme for 30 minutes, the outer membrane permeability increases by 3 times, creating conditions for Nisin penetration. Additionally, lysozyme activates the bacterial "cell wall repair stress response," consuming bacterial energy and indirectly reducing resistance.

(II) Nisin: "Second Step of Sterilization" to Act Precisely on Cell Membranes and Accelerate Cell Death

After lysozyme disrupts the cell wall barrier, Nisin can efficiently reach the bacterial cell membrane and exert its classic antimicrobial effects:

Nisin specifically binds to lipid II (a key precursor for bacterial cell wall synthesis) on the cell membrane, forming "Nisin-lipid II complexes." Multiple complexes aggregate and insert into the cell membrane, creating transmembrane pores with a diameter of 2–3 nm.

These pores drastically increase membrane permeability, causing a large leakage of intracellular small molecules (e.g., K⁺, amino acids) and allowing external water and harmful substances to enter the cell, ultimately leading to bacterial lysis and death. A study on Listeria monocytogenes (common in chilled ham) showed:

When Nisin (200 IU/g) is used alone, the bacteriostatic rate is only 65%;

When lysozyme (0.1%) is used alone, the bacteriostatic rate is 58%;

When combined, the bacteriostatic rate increases to 96%, and the bacterial death rate (log-phase killing time) shortens from 6 hours (single-treatment groups) to 2 hours, demonstrating a significant synergistic effect.

(III) Synergistic Enhancement: "Third Step" to Cover Broad-Spectrum Microorganisms and Inhibit Resistant Mutations

The microbial community in chilled meat products is complex (including Gram-positive bacteria, Gram-negative bacteria, and a small amount of fungi). A single antimicrobial agent often has an "antimicrobial spectrum blind spot," while the Nisin-lysozyme combination achieves complementary advantages:

Nisin compensates for lysozyme’s insufficient inhibitory ability against some Gram-positive bacteria (e.g., lysozyme has low hydrolysis efficiency for Staphylococcus aureus);

Lysozyme expands Nisin’s antimicrobial spectrum, inhibiting Gram-negative bacteria (e.g., Pseudomonas) that are insensitive to Nisin.

Furthermore, the combination reduces the risk of microbial resistant mutations: When Nisin is used alone, Listeria monocytogenes easily develops resistance by mutating the structure of lipid II (resistance rate ≈ 10⁻⁶). After combination, lysozyme breaks down the cell wall in advance, allowing Nisin to work without high concentrations, reducing the selection pressure for resistant mutations. Experiments confirm that after 10 consecutive generations of subculture, resistant strains appear in the single Nisin group, while no resistance is detected in the combined group, indicating better safety.

II. Efficacy Evaluation Dimensions of the Nisin-Lysozyme Combination in Chilled Meat Products

To evaluate the preservation efficacy of the combined system, a comprehensive evaluation framework must be established around three core dimensions—"microbial control," "quality maintenance," and "safety risk"—considering the storage characteristics of chilled meat products (primarily 4°C refrigeration):

(I) Microbial Indicators: Core Focus on "Antibacterial Efficiency" and "Shelf Life Extension"

Microbial count is a key indicator determining the spoilage of chilled meat products. The efficacy of the combined system must be verified through the following indicators:

Total Viable Count (TVC): The spoilage threshold for chilled meat products is typically TVC > 10⁶ CFU/g. For chilled ham without preservatives, TVC reaches 10⁷ CFU/g on the 10th day of 4°C refrigeration, accompanied by off-odors. For samples treated with Nisin (200 IU/g) + lysozyme (0.1%), TVC remains below 10⁶ CFU/g on the 25th day of 4°C refrigeration, extending the shelf life by 1.5 times.

Pathogen Control: Focus on pathogens such as Listeria monocytogenes and Staphylococcus aureus. For chilled sausages artificially contaminated with Listeria monocytogenes (initial concentration 10³ CFU/g), the concentration of Listeria monocytogenes in the combined treatment group drops to 10⁰ CFU/g (undetectable) on the 20th day of 4°C refrigeration, while the single Nisin group still has 10² CFU/g and the lysozyme group has 10¹ CFU/g, indicating a more thorough pathogen-killing effect in the combined group.

Inhibition of Dominant Spoilage Bacteria: Dominant spoilage bacteria in chilled meat products are mostly Pseudomonas (Gram-negative) and lactic acid bacteria (some cause acid-induced spoilage). The combined system achieves a 92% inhibition rate against Pseudomonas (vs. only 35% in the single Nisin group) and an 85% inhibition rate against acid-producing lactic acid bacteria, effectively preventing spoilage characteristics such as "sourness and stickiness."

(II) Quality Indicators: Key Focus on "Flavor, Color, and Texture" Maintenance

The consumer value of chilled meat products depends on quality stability. The combined system must control microorganisms without negatively affecting quality:

Sensory Flavor: Both Nisin and lysozyme are natural ingredients, free from the bitter or metallic taste of chemical preservatives (e.g., nitrite, potassium sorbate). Sensory evaluation (by a 10-person professional panel) shows that the flavor score (full score 10) of chilled ham treated with the combination decreases from an initial 9.2 to 8.5 within 20 days of 4°C refrigeration, significantly higher than the untreated group (score drops to 6.0 on the 10th day, with a rancid odor).

Color: The color of chilled meat products mainly depends on myoglobin. Microbial spoilage causes myoglobin oxidation, darkening the meat color. The redness value (a-value, measuring meat color vividness) of the combined treatment group decreases from an initial 18.5 to 16.2 within 25 days of refrigeration, while the untreated group’s a-value drops to 12.3 on the 10th day, showing graying.

Texture Characteristics: Microbial decomposition of proteins causes meat products to soften and lose elasticity. The hardness value (reflecting texture firmness) of chilled sausages treated with the combination decreases from an initial 2500 g to 2300 g within 20 days of refrigeration, and the elasticity value decreases from 0.9 to 0.85. In contrast, the untreated group’s hardness value drops to 1800 g and elasticity value to 0.6 on the 10th day, showing obvious texture deterioration.

(III) Safety Indicators: Core Focus on "No Residues and No Toxicity"

Chilled meat products are consumed directly, so the safety of the combined system must be verified through residue, toxicity, and nutrient retention tests:

Residue Detection: Nisin and lysozyme can be decomposed into amino acids by digestive enzymes in the human intestine, with no residue risk. High-performance liquid chromatography (HPLC) detection shows that the residue of Nisin in chilled ham treated with the combination remains stable at 180–200 IU/g throughout refrigeration (complying with the maximum usage limit of 3000 IU/g specified in GB 2760), and lysozyme residue is < 0.1% (within the safe range), with no over-limit risk.

Acute Toxicity Test: In oral acute toxicity tests on mice, the median lethal dose (LD₅₀) of the Nisin-lysozyme combination is > 5000 mg/kg, classified as "practically non-toxic," far higher than the estimated daily intake for humans (approximately 0.1 mg/kg body weight).

Nutrient Retention Rate: The combined system does not affect nutrients such as proteins and vitamins in meat products. Detection shows that soy-braised beef treated with the combination retains 95% of its protein and 88% of B vitamins within 20 days of refrigeration, with no significant difference from the untreated group (93% protein retention, 85% B vitamin retention), resulting in minimal nutrient loss.

III. Application Optimization of the Combined System in Chilled Meat Products: Concentration, Process, and Storage Adaptation

To maximize the preservation efficacy of the combined system, application schemes must be optimized based on the processing technology (e.g., injection, tumbling, enema) and storage conditions of chilled meat products:

(I) Concentration Optimization: Balancing Efficacy and Cost

Excessively high concentrations increase costs and may affect taste, so the optimal ratio must be determined through orthogonal experiments:

Chilled Ham: The optimal ratio is Nisin 200 IU/g + lysozyme 0.1%—this concentration achieves the best TVC control effect, and the cost is 20% lower than using Nisin alone (300 IU/g).

Chilled Sausages: Due to their higher fat content (easily decomposed by microorganisms), concentrations need to be moderately increased. The optimal ratio is Nisin 250 IU/g + lysozyme 0.12%, which effectively inhibits fat-decomposing bacteria (e.g., Pseudomonas).

Soy-Braised Beef: With high water content (prone to microbial growth), the optimal ratio is Nisin 300 IU/g + lysozyme 0.15%, extending the refrigerated shelf life to 30 days.

(II) Process Adaptation: Ensuring Uniform Distribution

The processing technology of chilled meat products affects the uniform distribution of the combined system, so the addition method must be adjusted accordingly:

Injection Process (e.g., chilled ham): Dissolve Nisin and lysozyme in brine, then inject into meat chunks using an injection machine (pressure 0.3 MPa) to ensure the antimicrobial agents penetrate into the muscle tissue (avoiding surface-only distribution, which causes "internal spoilage").

Tumbling Process (e.g., sausage filling): Add the combined system during the tumbling stage (4°C, rotation speed 10 r/min, duration 2 hours). Tumbling ensures full mixing of the antimicrobial agents with the meat filling, increasing distribution uniformity by 40%.

Post-Ripening Process: After adding the combined system, control the post-ripening temperature (60–65°C, duration 30 minutes) to avoid Nisin and lysozyme inactivation due to high temperatures. Experiments show that at temperatures below 65°C, the activity retention rate of both is > 90%; at temperatures above 70°C, the retention rate drops to 70%.

(III) Storage Adaptation: Combining with Cold Chain Control

The combined system must be used with a cold chain to maximize shelf life extension:

Temperature Control: 4°C refrigeration is the optimal storage temperature, as the antimicrobial activity of the combined system is stable at this temperature. If the temperature rises to 10°C (cold chain breakage), the preservation efficacy of the combined system decreases by 30%, requiring a shortened shelf life (from 25 days to 15 days).

Packaging Adaptation: Vacuum packaging (oxygen concentration < 1%) reduces oxidative spoilage and the growth of aerobic bacteria (e.g., Pseudomonas), synergizing with the combined system. Chilled ham treated with vacuum packaging + the combination has a 5-day longer shelf life than that treated with ordinary packaging + the combination, achieving better results.

Through the mechanism of "lysozyme cell wall breakdown – Nisin sterilization – synergistic broad-spectrum antimicrobial activity," the Nisin-lysozyme combination achieves "efficient microbial control, stable quality, and non-toxicity" in chilled meat products. Under 4°C refrigeration, it extends the shelf life of products such as chilled ham and sausages from 10–15 days to 25–30 days, while effectively controlling pathogens like Listeria monocytogenes and maintaining the flavor, color, and texture of meat products. Its advantages—naturalness, no residues, broad antimicrobial spectrum, and no resistance risk—align with consumer demand for "clean label" foods and provide a feasible path for the chilled meat product industry to reduce reliance on chemical preservatives.

Future research can focus on three areas: developing microcapsule embedding technology for the combined system (to improve high-temperature stability), combining it with natural plant extracts (e.g., tea polyphenols) to further enhance preservation efficacy, and promoting the application of combined preservation technology in more categories of chilled meat products.