The Application of Nisin in Seafood Products

As a natural antimicrobial peptide produced by the fermentation of Streptococcus lactis,nisin boasts advantages such as safety (non-toxicity), precise antibacterial spectrum (primarily targeting Gram-positive bacteria), high temperature resistance, and easy degradability. It has been widely applied in the field of food preservation. Seafood products (especially fish and shrimp) are prone to microbial contamination and spoilage due to their high moisture content, rich protein, and near-neutral pH. Among the causative microorganisms, Gram-positive spoilage bacteria (e.g., Listeria monocytogenes, Bacillus cereus) and some "Gram-positive-Gram-negative spoilage bacterial complexes" are the core flora triggering quality deterioration. By damaging bacterial cell membranes and inhibiting spore germination, nisin can specifically block the growth and reproduction of spoilage bacteria in fish and shrimp, extend product shelf life, while preserving the natural flavor and nutritional value of seafood—providing a green solution for the preservation of seafood products.

I. Main Spoilage Bacteria in Fish and Shrimp and Their Hazards: Targets of nisin

During fishing, transportation, and processing, fish and shrimp are easily contaminated by environmental microorganisms. Among these, aerobic and facultatively anaerobic Gram-positive bacteria are the main contributors to spoilage. Their metabolic activities produce off-flavor substances and decompose nutrients, seriously affecting product quality and food safety.

(I) Core Spoilage Bacteria in Fish Products

Listeria monocytogenesAs a typical Gram-positive foodborne pathogen, it can survive and reproduce at low temperatures (0–4°C), making it a major risk bacterium in refrigerated fish products (e.g., cold-smoked salmon, ready-to-eat cod fillets). It not only causes spoilage symptoms such as increased mucus and sticky flesh in fish products but also may induce human food poisoning (e.g., fever, diarrhea, meningitis), posing significant risks to immunocompromised populations such as pregnant women and the elderly.

Bacillus cereusWidely present on the surface and in the intestines of freshwater fish (e.g., grass carp, crucian carp), it produces heat-resistant spores that cannot be completely eliminated during the heating process of processed products like dried fish and fish balls. After cooling, the spores germinate into vegetative cells, which metabolize to produce emetic toxins and diarrheal toxins. This causes the products to develop a rancid taste and loose flesh within 1–2 days of storage, while also triggering food poisoning.

Lactobacillus (some spoilage strains)Although Lactobacillus is beneficial in fermented foods, some strains (e.g., specific strains of Lactobacillus plantarum) over-reproduce in cold-stored white fish (e.g., cod, flounder). They decompose proteins and nucleotides in fish meat, producing off-flavor substances such as trimethylamine and histamine, leading to a "fishy odor" and rapid quality deterioration of the fish.

(II) Core Spoilage Bacteria in Shrimp Products

Staphylococcus aureusCommonly found on the shells and gills of fresh shrimp, it multiplies rapidly at room temperature (20–30°C) and metabolizes to produce heat-resistant enterotoxins (e.g., type A and B toxins). These toxins cannot be destroyed even by subsequent heating; consumption may cause poisoning symptoms such as vomiting and abdominal pain. This bacterium reproduces faster in semi-dried products like dried shrimp and salted shrimp, causing product spoilage within 3–5 days.

Bacillus (e.g., Bacillus subtilis)Widely present in the marine environment, it easily attaches to the surface of shrimp. The spores it produces can withstand the cooking process (80–100°C for 10 minutes) during shrimp product processing. After the product cools, the spores germinate, decomposing proteins and polysaccharides in shrimp meat to produce mucus and a putrid odor. This causes quality deterioration of quick-frozen products like shrimp balls and shrimp paste after thawing.

Gram-positive-Gram-negative spoilage bacterial complexIn the early stage of fresh shrimp spoilage, Gram-positive bacteria (e.g., Staphylococcus) first decompose small-molecule carbohydrates in shrimp meat, lowering the environmental pH and creating conditions for the subsequent reproduction of Gram-negative bacteria (e.g., Vibrio parahaemolyticus), forming a "synergistic spoilage" effect. Although nisin has weak direct inhibitory effects on Gram-negative bacteria, it can block this "synergistic effect" by inhibiting the initial reproduction of Gram-positive bacteria, indirectly delaying the overall spoilage process.

II. Mechanism of Nisin Inhibiting Spoilage Bacteria in Fish and Shrimp: Precise Targeting of Bacterial Structure and Metabolism

Nisin exhibits clear targeting in its antibacterial action, primarily by damaging the cell membrane integrity of Gram-positive bacteria and inhibiting spore germination—blocking spoilage bacteria growth from both the "reproductive stage" and "dormant stage". Its mechanism involves three core links:

(I) Damaging Bacterial Cell Membranes and Causing Content Leakage

Gram-positive bacteria have only a peptidoglycan layer outside their cell membranes (no outer membrane barrier). Nisin can specifically bind to "lipid II" (a key precursor for bacterial cell wall synthesis) on the cell membrane via the lanthionine residues in its molecular structure, forming a "nisin-lipid II complex". This complex accumulates on the cell membrane, forming transmembrane channels with a diameter of approximately 2–3 nm. This increases membrane permeability, causing a large leakage of small-molecule substances (e.g., potassium ions, amino acids, ATP) from the cell, while external water and harmful substances enter the cell. Ultimately, this leads to bacterial osmotic imbalance and metabolic disorders, resulting in cell death. For Listeria monocytogenes and Staphylococcus aureus in fish and shrimp, the minimum inhibitory concentration (MIC) of nisin is only 0.125–0.5 μg/mL, far lower than the allowable addition level of food-grade nisin (GB 2760 stipulates a maximum addition of 0.2 g/kg), enabling rapid inhibition of these spoilage bacteria at low doses.

(II) Inhibiting Spore Germination and Blocking the "Dormancy-Reproduction" Cycle

Bacteria such as Bacillus cereus and Bacillus subtilis in fish and shrimp produce heat-resistant spores that cannot be completely eliminated by conventional heating. Nisin inhibits spore germination through two methods:

Binding to the spore cortex: Nisin can penetrate the spore coat and cortex, binding to peptidoglycan in the cortex to prevent cortex degradation (a key step in spore germination), thereby preventing the spore from releasing vegetative cells.

Interfering with germination signals: Nisin can competitively bind to "germination receptors" on the spore surface, blocking the binding of germination signal molecules (e.g., amino acids, carbohydrates), keeping the spore in a dormant state for a long time and preventing it from reproducing into spoilage-causing vegetative cells.

Experiments show that adding 0.1 g/kg nisin to shrimp ball processing can reduce the spore germination rate of Bacillus subtilis by more than 80%, extending the shelf life of the product from 7 days to 15 days under 4°C refrigeration.

(III) Synergizing with Other Factors to Enhance Antibacterial Effects

In the complex system of fish and shrimp products, nisin can synergize with environmental factors (e.g., pH, salt content) and processing techniques (e.g., low temperature, mild heating) to further enhance its inhibitory effects on spoilage bacteria:

Low pH synergy: Under mild acidification of fish and shrimp products (e.g., adding 0.1% citric acid), the antibacterial activity of nisin can be enhanced by 2–3 times. Low pH increases membrane permeability, promoting the binding of nisin to lipid II and accelerating bacterial death.

Salt synergy: Sodium chloride (3%–5%) in salted fish and shrimp products damages the stability of bacterial cell membranes, forming a "dual membrane damage" effect with nisin, significantly reducing the drug resistance of spoilage bacteria.

Low temperature synergy: Refrigeration (0–4°C) slows bacterial metabolism, giving nisin more time to bind to bacterial cell membranes and extending its antibacterial action time—especially suitable for preserving ready-to-eat fish and shrimp products.

III. Specific Application Methods of nisin in Fish and Shrimp Products: Adapting to Different Processing Scenarios

Based on the type of fish and shrimp products (fresh products, processed products, quick-frozen products) and processing techniques, the application method of nisin should be adjusted to ensure uniform distribution, sufficient contact with spoilage bacteria, and no impact on product taste or appearance.

(I) Pretreatment Preservation of Fresh Fish and Shrimp

Fresh fish and shrimp spoil quickly after fishing; Nisin can be added via "soaking" or "spraying" to extend transportation and storage periods:

Soaking method: Soak fresh fish and shrimp (e.g., salmon, prawns) in a preservative solution containing nisin (50–100 μg/mL), along with 0.1% vitamin C (to prevent oxidative discoloration) and 0.5% potassium sorbate (synergistic antibacterial). The soaking time is 10–15 minutes (adjusted based on the size of the fish/shrimp). After soaking, drain and refrigerate (0–4°C). This method can extend the shelf life of fresh salmon from 3 days to 7 days and that of prawns from 2 days to 5 days, without affecting the elasticity of fish meat or the umami of shrimp.

Spraying method: For large fish (e.g., tuna, cod) or batch-processed shrimp, use spray equipment to evenly spray a nisin preservative solution (80–120 μg/mL) onto the product surface, with a spray volume of approximately 50 mL per square meter. After spraying, wrap with plastic wrap and refrigerate. This method is easy to operate, suitable for assembly line operations in aquatic processing plants, and effectively inhibits Staphylococcus and Bacillus on the surface of fish and shrimp.

(II) Preservation of Processed Fish and Shrimp Products

Processed products such as dried fish, fish balls, and shrimp paste are prone to cross-contamination during production. Nisin can be integrated into the product system or attached to the surface via "direct addition" or "coating":

Direct addition method: During the grinding stage of raw materials for fish balls and shrimp paste, add nisin (0.05–0.1 g/kg, based on total raw material weight) along with other auxiliary materials (e.g., starch, salt, monosodium glutamate), and stir evenly (300–500 rpm for 5–10 minutes) to ensure uniform distribution of nisin in the meat paste. This method inhibits Bacillus cereus introduced during processing, extending the shelf life of fish balls from 6 months to 9 months under -18°C freezing, with no mucus or off-flavors after thawing.

Coating method: For semi-dried products such as dried fish and salted shrimp, adopt a composite preservation technology of "edible coating + Nisin". Prepare a coating solution by mixing nisin (0.1–0.2 g/kg) with chitosan (1%–2%) or sodium alginate (0.5%–1%). Soak or brush the dried fish/shrimp with the coating solution, then dry (40–50°C for 2–3 hours) to form a transparent antibacterial coating. This coating not only blocks contact between oxygen and microorganisms but also slowly releases nisin to continuously inhibit the growth of Staphylococcus aureus, extending the shelf life of dried fish from 15 days to 30 days.

(III) Safety Protection of Ready-to-Eat Fish and Shrimp Products

Ready-to-eat fish and shrimp products (e.g., cold-smoked salmon, ready-to-eat shrimp meat) are consumed directly, requiring high microbial safety. A combined process of "Nisin + mild sterilization" is needed to achieve dual protection of "preservation + safety":

Cold-smoked salmon: Before smoking, soak salmon fillets in a mixed solution of nisin (100 μg/mL) and lactic acid (0.2%) for 20 minutes, then vacuum-pack (vacuum degree > -0.09 MPa) after smoking. Nisin inhibits Listeria monocytogenes that may contaminate during cold smoking, extending the shelf life of the product from 14 days to 28 days under 4°C refrigeration, without affecting the smoked flavor.

Ready-to-eat shrimp meat: Soak cooked and cooled shrimp meat in a preservative solution of nisin (80 μg/mL) and tea polyphenols (0.1%) for 10 minutes, drain, vacuum-pack, and pasteurize at 60°C for 15 minutes. This process completely eliminates Staphylococcus aureus in the shrimp meat, while nisin inhibits the germination of any remaining spores after sterilization. The product has a shelf life of up to 6 months at room temperature (25°C), suitable for room-temperature circulation.

IV. Precautions for Nisin Application: Ensuring Safety and Effectiveness

When applying nisin in fish and shrimp products, attention should be paid to three key points: "dosage control, compatibility taboos，and process adaptation" to avoid reduced antibacterial effects or impaired product quality due to improper use.

(I) Strictly Control Addition Dosage to Comply with Safety Standards

According to GB 2760 National Food Safety Standard for the Use of Food Additives, the maximum dosage of nisin in aquatic products is 0.2 g/kg (based on pure product). In practice, adjust the dosage based on product type and spoilage risk:

Pretreatment of fresh fish and shrimp: Recommended dosage is 50–120 μg/mL (concentration of soaking/spraying solution), equivalent to approximately 0.03–0.08 g/kg of raw material addition—far below the safety limit.

Processed products: Recommended addition for fish balls and shrimp paste is 0.05–0.1 g/kg; for semi-dried products such as dried fish and salted shrimp, the recommended coating solution concentration is 0.1–0.2 g/kg (equivalent to approximately 0.08–0.15 g/kg of raw material addition). Avoid excessive addition, as it may cause a slight bitter taste (nisin naturally has a weak bitter taste, which becomes noticeable when overused).

(II) Avoid Compatibility Conflicts to Prevent Reduced Antibacterial Activity

The antibacterial activity of Nisin is easily affected by certain components; note the following during application:

Avoid simultaneous use with proteases: If proteases (e.g., papain, used for tenderizing meat) are added to fish and shrimp products, they will degrade the peptide chain structure of Nisin, rendering it inactive. Add Nisin only after the protease has finished acting (e.g., 2 hours after tenderization).

Control metal ion concentration: High concentrations of calcium ions (>0.1%) and magnesium ions (>0.05%) can bind to Nisin, forming insoluble complexes that reduce its solubility and antibacterial activity. When adding Nisin, control the dosage of calcium and magnesium salts, or add nisin before adding metal ions.

Avoid mixing with strong alkaline substances: Nisin is prone to denaturation and inactivation in an alkaline environment with pH > 8.0. The pH of fish and shrimp products is usually 6.0–7.5 (near neutral), requiring no additional adjustment. However, avoid direct mixing with strong alkaline auxiliary materials such as sodium bicarbonate; add them in stages (first add alkaline auxiliary materials to adjust pH, then add nisin).

(III) Adapt to Processing Techniques to Ensure Uniform Distribution

Different processing techniques significantly affect the stability and uniform distribution of nisin; adjust accordingly:

Adaptation to heating processes: Nisin has good heat resistance (retaining over 80% activity after sterilization at 121°C for 30 minutes) and can withstand cooking (100°C for 10 minutes) and baking (180°C for 5 minutes) of fish and shrimp products. However, avoid prolonged contact with raw materials at high temperatures (>121°C). It is recommended to add nisin 1–2 hours before heating, or during the cooling stage after heating (<60°C) (e.g., soaking ready-to-eat shrimp meat after pasteurization).

Homogenization and stirring: For minced meat products such as fish balls and shrimp paste, ensure uniform distribution of nisin through sufficient stirring (300–500 rpm for 5–10 minutes) or homogenization (20–30 MPa), avoiding uneven antibacterial effects due to local low concentrations.

Packaging and storage: Fish and shrimp products with added nisin should be vacuum-packaged or nitrogen-packaged to reduce oxygen contact (Nisin is easily oxidized and degraded in an aerobic environment). Meanwhile, store in a low-temperature environment (0–4°C refrigeration or -18°C freezing) to further extend shelf life.

As a natural antimicrobial peptide, nisin exhibits clear inhibitory effects on spoilage bacteria in fish and shrimp products. It can specifically block the growth of core spoilage bacteria such as Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus through a dual mechanism of "damaging cell membranes + inhibiting spore germination". Combined with flexible application methods (soaking, spraying, direct addition, coating), it adapts to different scenarios such as fresh fish/shrimp pretreatment, processed product preservation, and ready-to-eat product safety protection—extending shelf life while preserving the natural flavor and nutritional value of seafood. During application, strictly control dosage, avoid compatibility conflicts, and adapt to processing techniques to ensure safety and effectiveness. Compared with chemical preservatives (e.g., sodium benzoate, potassium sorbate), nisin has the advantages of "natural origin, safety, and no residues", meeting consumer demand for green food and holding broad application prospects in the preservation of seafood products in the future.