Precautions for the combined use of Nisin and chitosan

Nisin (a natural bacteriocin) exhibits potent inhibitory effects against Gram-positive bacteria (e.g., Staphylococcus, Streptococcus). Chitosan, a deacetylated derivative of chitin, integrates multiple functions including broad-spectrum antimicrobial activity (against bacteria and fungi), film-forming ability, and preservative properties. When used in combination, they enhance application efficacy through synergistic antimicrobial effects and functional complementarity—for instance, chitosan broadens Nisin’s narrow antimicrobial spectrum, while chitosan’s film-forming property extends Nisin’s duration of action. This combination is widely applied in food preservation (e.g., meat products, dairy products, fruit and vegetable freshness retention) and biomedical materials (e.g., wound dressings).

However, challenges during compounding—such as differences in physicochemical properties, application scenario limitations, and fluctuating synergistic effects—often lead to reduced antimicrobial efficiency, decreased stability, and impaired product quality. Clarifying core considerations for their combined use is critical to unlocking synergistic advantages and mitigating risks. This article systematically outlines key precautions and practical recommendations across four dimensions: physicochemical control of the compound system, application scenario adaptation, stability maintenance, and safety assurance.

I. Physicochemical Control of the Compound System: Preventing Synergy Failure

The synergistic effect of Nisin and chitosan depends on their uniform dispersion and benign intermolecular interactions in the system. Physicochemical factors such as pH, ionic strength, and compound ratio directly affect their solubility, charge state, and antimicrobial activity, requiring precise control.

(I) pH Adjustment: Matching the Optimal Activity Range for Both

Both Nisin and chitosan exhibit pH-dependent antimicrobial activity. The compound system must balance their optimal pH ranges to avoid activity loss due to improper pH:

pH characteristics of Nisin: As an acidic polypeptide (isoelectric point pI ≈ 5.0), Nisin maintains high stability and antimicrobial activity in acidic environments (pH 2.0–6.0). Acidic conditions preserve its molecular structure and prevent peptide bond hydrolysis. At pH > 7.0, Nisin is easily degraded by alkaline proteases, losing over 50% of its activity within 24 hours.

pH characteristics of chitosan: Chitosan contains amino groups (-NH₂) and only dissolves and exerts antimicrobial effects in acidic environments (pH 4.0–6.0), where amino groups protonate into positively charged -NH₃⁺. These positive charges adsorb to negatively charged bacterial cell membranes, disrupting membrane structure. At pH > 6.5, chitosan precipitates due to deprotonation, losing solubility and antimicrobial activity.

Optimal pH for the compound system: Integrating both properties, the pH of the compound system should be controlled between 4.0–5.5. Within this range, Nisin remains structurally stable, chitosan dissolves completely, and their "charge synergy" (weak negative charge of Nisin and positive charge of chitosan) promotes uniform dispersion and enhances antimicrobial efficacy.

In practice: Avoid strong acids (e.g., hydrochloric acid) or alkalis (e.g., sodium hydroxide) for pH adjustment. Use citrate-sodium citrate buffer or acetate-sodium acetate buffer instead, and adjust to the target pH gradually (stir for 10–15 minutes after each adjustment to ensure system uniformity) to prevent local pH fluctuations that cause precipitation of Nisin or chitosan.

(II) Ionic Strength Control: Avoiding Salt Ion Interference with Dispersion and Activity

Salt ions (e.g., Na⁺, Cl⁻, Ca²⁺) in the system disrupt the solubility and intermolecular interactions of Nisin and chitosan via "charge shielding effects," requiring strict control of ionic strength:

Impact on chitosan: Chitosan dissolution relies on hydrogen bonding between protonated amino groups and water molecules. High-concentration salt ions (e.g., NaCl > 0.5 mol/L) compete with chitosan for water molecules, reducing solubility and even causing flocculation. Additionally, Cl⁻ shields chitosan’s positive charge, weakening its adsorption to bacterial cell membranes and lowering antimicrobial activity.

Impact on Nisin: Nisin’s antimicrobial activity depends on its binding to lipid carriers on bacterial cell membranes. High-concentration divalent ions (e.g., Ca²⁺, Mg²⁺) compete with Nisin for binding sites, inhibiting membrane disruption. Salt ions may also accelerate Nisin hydrolysis, shortening its shelf life.

Control strategy: The ionic strength of the compound system must be < 0.1 mol/L (equivalent to NaCl concentration < 0.58%). In practical applications:

If raw materials (e.g., food matrices, culture media) contain high salt levels, pre-test salt concentrations.

If salt concentrations exceed limits, use "dilution" (dilute raw materials with deionized water to < 0.5% salt) or "dialysis" (use dialysis bags with 3000 Da molecular weight cutoff to remove excess salt ions).

Avoid high-salt additives (e.g., table salt, phosphates) during compounding. For flavoring or water retention, choose low-salt alternatives (e.g., low-sodium salt, trehalose).

(III) Compound Ratio Optimization: Balancing Synergistic Antimicrobial Efficacy and Cost-Efficiency

The compound ratio of Nisin to chitosan directly affects synergistic effects. Improper ratios may cause "synergy failure" (e.g., excess chitosan masking Nisin’s active sites) or "high costs" (Nisin is far more expensive than chitosan). Optimal ratios must be determined via pre-experiments:

Basic ratio range: Depending on application scenarios, the mass ratio is typically controlled between 1:10–1:100 (1 part Nisin to 10–100 parts chitosan):

In food preservation: Nisin is usually added at 0.01–0.05 g/kg, with corresponding chitosan addition of 0.1–5 g/kg.

In biomedical materials (e.g., wound dressings): Nisin concentration is 0.1–0.5 g/L, with chitosan concentration of 1–10 g/L.

Ratio optimization method: Use the "checkerboard method" to test different compound ratios (e.g., 1:10, 1:20, 1:50, 1:100), determine the minimum inhibitory concentration (MIC) against target microorganisms (e.g., Staphylococcus aureus in food, Penicillium in fruits/vegetables), and select the ratio with "lowest MIC and lowest cost." For example, against Listeria in dairy products, a 1:20 ratio may reduce the MIC by 40% compared to Nisin alone and 60% compared to chitosan alone, achieving optimal synergy.

Precautions:

Avoid excessively high Nisin ratios (e.g., > 1:5), as this increases costs and causes Nisin aggregation, reducing contact efficiency with bacteria.

Avoid excessively high chitosan ratios (e.g., > 1:200), as excess chitosan forms dense films that block Nisin diffusion to microorganisms, weakening antimicrobial effects.

II. Application Scenario Adaptation: Preventing Efficacy Attenuation from Matrix Interactions

The efficacy of the Nisin-chitosan combination is significantly influenced by application scenarios (e.g., food matrices, material types). Components in different matrices (e.g., proteins, fats, polysaccharides) may interact with Nisin or chitosan, reducing activity or impairing product quality. Usage methods must be adjusted accordingly.

(I) Food Industry Applications: Adapting to Matrix Characteristics

Food matrices are complex (e.g., meat products are high in protein/fat; fruits/vegetables contain organic acids and polyphenols). Compound protocols must be adjusted based on matrix properties:

1. High-Protein/High-Fat Foods (e.g., Meat Products, Dairy Products)

Risks:

Proteins (e.g., casein, myofibrillar proteins) bind to Nisin, forming complexes that mask its active sites.

Fats hinder uniform dispersion of Nisin and chitosan, reducing antimicrobial efficiency.

Countermeasures:

Pre-form "nanocomposites" of Nisin and chitosan (via ultrasonic treatment or emulsifier assistance to form 100–500 nm particles) to reduce non-specific binding to proteins/fats.

Adjust addition timing: Add during late-stage processing (e.g., after meat 灌肠，before dairy pasteurization) to avoid Nisin inactivation by high-temperature processes (e.g., boiling, frying).

Control water activity (Aw < 0.9): Low Aw reduces protein-Nisin interactions and extends chitosan’s film-forming preservation period.

2. High-Acid Foods (e.g., Juices, Pickles, pH < 4.0)

Risks:

While acidic environments stabilize Nisin, excessive acidity (e.g., pH < 3.0) causes over-protonation of chitosan, leading to molecular chain contraction and reduced film-forming/antimicrobial activity.

Polyphenols in juices form hydrogen bonds with chitosan, causing precipitation.

Countermeasures:

Adjust food pH to 4.0–4.5 using buffers (e.g., adding small amounts of sodium citrate to juices) to balance activity of both components.

Select low-deacetylation chitosan (70%–80% deacetylation): It exhibits more stable solubility in high-acid environments and less binding to polyphenols.

Use "spraying" instead of "addition": Apply the compound solution directly to food surfaces (e.g., pickle jar mouths, juice packaging interiors) to minimize direct contact with the food matrix.

(II) Biomedical Material Applications: Balancing Biocompatibility and Activity

In biomedical materials (e.g., wound dressings, tissue engineering scaffolds), the compound system must simultaneously meet "antimicrobial activity" and "biocompatibility" (no cytotoxicity, no irritation). Key considerations include:

1. Cytotoxicity Control

Risks:

High-concentration chitosan (> 20 g/L) excessively adsorbs to cell surfaces via positive charges, inhibiting cell proliferation.

While Nisin has low toxicity to human cells, excess concentrations (> 1 g/L) may trigger local inflammatory responses.

Countermeasures:

Control concentrations: Nisin at 0.1–0.5 g/L, chitosan at 1–5 g/L. Verify via MTT assay that the survival rate of fibroblasts (key cells for wound repair) exceeds 90%.

Select low-molecular-weight chitosan (< 50 kDa): It has good solubility, low cytotoxicity, and degrades easily into absorbable glucosamine, avoiding foreign body reactions from long-term retention.

2. Balancing Degradability and Stability

Risks:

Chitosan degrades easily in vivo via lysozymes. Rapid degradation causes premature Nisin release, shortening antimicrobial duration; slow degradation may impair wound healing.

Countermeasures:

"Crosslinking modification": Mildly crosslink chitosan with glutaraldehyde (crosslinking degree < 5%) to slow degradation, matching Nisin release to wound healing cycles (typically 7–14 days).

"Layered loading technology": Load Nisin on the chitosan film surface (rapid release for early infection control) and small amounts in the chitosan matrix (sustained release for long-term antimicrobial activity), achieving "immediate + prolonged" efficacy.

III. Stability Maintenance of the Compound System: Extending Shelf Life and Activity Duration

The Nisin-chitosan compound system is prone to activity degradation from temperature, light, and oxygen during storage and use. Stability must be enhanced through storage condition control and modification to extend activity duration.

(I) Storage Condition Control: Mitigating Temperature, Light, and Oxygen Effects

Temperature control: Nisin is sensitive to high temperatures (80% activity loss after 30 minutes at > 80℃), and chitosan undergoes thermal degradation at high temperatures. Store the compound system at 4–25℃ (≤ 3 months at room temperature, ≤ 6 months at 4℃ refrigeration). Avoid repeated freeze-thaw cycles (freezing causes chitosan precipitation, which cannot be redissolved after thawing). For long-term storage, lyophilize the compound solution (-50℃ to -40℃, vacuum < 10 Pa) and reconstitute with sterile deionized water before use—activity loss can be controlled below 10%.

Light control: Ultraviolet light damages Nisin’s peptide bonds and accelerates chitosan oxidation. Package the compound system in brown glass bottles or opaque plastic containers, and store in cool, dark environments (e.g., light-proof cabinets) to avoid direct sunlight or strong light (e.g., laboratory UV lamps, intense light in food processing workshops).

Oxygen control: Oxygen oxidizes Nisin’s thiol groups (-SH) and chitosan’s amino groups, reducing activity. Flush packaging with food-grade nitrogen (> 99.9% purity) or add small amounts of natural antioxidants (e.g., vitamin E, tea polyphenols, concentration < 0.1%) to minimize oxidation.

(II) Stability Modification: Enhancing Degradation Resistance via Modification

Address stability limitations of the compound system by modifying chitosan to indirectly improve Nisin stability:

Hydrophobic modification: Esterify chitosan with fatty acids (e.g., caprylic acid, lauric acid) to enhance hydrophobicity, reducing Nisin peptide bond hydrolysis by water molecules. Hydrophobically modified chitosan also forms denser films, blocking oxygen-Nisin contact and extending activity duration.

Nanoencapsulation: Embed Nisin in chitosan nanoparticles (50–200 nm). Chitosan nanoparticles act as "protective shells," preventing Nisin degradation by enzymes or binding to matrix components. Additionally, nanoparticles enable "sustained release" of Nisin, prolonging antimicrobial activity (e.g., in fruit/vegetable preservation, antimicrobial duration extends from 7 days to 14 days).

IV. Safety Assurance: Mitigating Potential Risks and Ensuring Compliance

While Nisin and chitosan are generally recognized as safe natural substances, their combined use requires attention to residues, allergy risks, and compliance to meet relevant standards.

(I) Residue Control: Meeting Food and Medical Standards

Food industry: Nisin’s maximum usage in food must comply with GB 2760 National Food Safety Standard for the Use of Food Additives (e.g., ≤ 0.5 g/kg in meat products, ≤ 0.3 g/kg in dairy products). Chitosan must meet GB 1886.317 National Food Safety Standard for Food Additive Chitosan (purity ≥ 90%, deacetylation ≥ 75%). When used in combination, calculate the actual addition of each component separately to avoid exceeding limits (e.g., if Nisin already reaches 0.5 g/kg, no additional Nisin should be added—only adjust chitosan ratio).

Medical industry: Chitosan must comply with YY/T 0453 Medical Chitosan Materials (cytotoxicity ≤ Grade 1, no sensitization). Nisin must meet pharmaceutical-grade standards (purity ≥ 95%, heavy metals < 10 ppm). Compound materials must pass animal skin irritation tests (e.g., rabbit skin tests with no redness or exudation) and allergy tests (e.g., guinea pig maximization tests with no allergic reactions) to ensure clinical safety.

(II) Allergy Risk Mitigation: Focus on Special Populations and Cross-Allergies

Allergy screening:

Nisin is derived from lactic acid bacteria; individuals allergic to lactic acid bacteria (e.g., rash, diarrhea after consuming yogurt) may be allergic to Nisin and should avoid food or medical materials containing the compound system.

Chitosan is derived from crustaceans (e.g., shrimp, crabs); individuals allergic to seafood may be allergic to chitosan. Plant-sourced chitosan (e.g., fungal chitosan) can be used as a substitute to reduce allergy risks.

Cross-allergy testing: Before using biomedical materials (e.g., wound dressings), perform local skin tests on patients (apply a small amount of the compound solution to the inner forearm and observe for 24 hours—no redness or itching indicates a negative result) to avoid allergic reactions from direct use.

(III) Compliance Verification: Meeting Regional and Industry Standards

Standards for Nisin and chitosan vary by country/region; compliance must be verified in advance:

In the EU: Nisin is designated as food additive E234, and chitosan must comply with EC No. 1333/2008.

In the US: The FDA classifies Nisin as "Generally Recognized as Safe" (GRAS), and chitosan requires FDA Drug Master File (DMF) registration.

Manufacturers must provide raw material certification (e.g., purity test reports, microbial limit reports), and compound products must pass third-party testing (e.g., antimicrobial activity tests, heavy metal tests) to meet target market standards.

The combined use of Nisin and chitosan must focus on "maximizing synergistic activity and minimizing risks." Core considerations include:

Ensuring synergistic antimicrobial efficacy via precise control of pH (4.0–5.5), ionic strength (< 0.1 mol/L), and compound ratio (1:10–1:100).

Adjusting compound protocols based on application scenarios (food matrices, medical materials) to avoid activity attenuation from matrix interactions.

Extending system stability through optimized storage conditions (low temperature, dark, oxygen-free) and modification.

Ensuring safe application by monitoring residues, mitigating allergy risks, and verifying compliance.

Only by fully understanding the physicochemical properties and application boundaries of Nisin and chitosan can scientific compounding unlock their "1+1 > 2" synergistic advantages, providing efficient, safe solutions for food preservation, biomedicine, and other fields.