The antibacterial effect of Nisin in the preservation of soaked chicken feet

Pickled chicken feet, a typical low-temperature meat product (usually refrigerated at 0–4℃), are highly favored by consumers for their spicy-sour taste and chewy texture. However, during processing, they are susceptible to quality deterioration due to raw material contamination (e.g., Gram-positive bacteria on chicken feet), microbial growth in processing environments (e.g., Staphylococcus residues on equipment), and psychrophilic bacterial proliferation during low-temperature storage. These issues lead to spoilage—characterized by bag swelling, off-odors, and soft texture—and shortened shelf life.

Nisin (nisin), a natural bacteriocin, exhibits potent inhibitory effects against the main spoilage bacteria in pickled chicken feet (such as Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus). It also aligns with consumer demand for "clean labels," making it an ideal antibacterial agent for preserving pickled chicken feet. A thorough analysis of its antibacterial mechanisms, application effects, and influencing factors is of great significance for extending product shelf life and ensuring food safety. This article systematically elaborates on the value and practical key points of Nisin in preserving pickled chicken feet from four dimensions: antibacterial targets, practical application effects, key influencing factors, and optimization strategies.

I. Core Antibacterial Mechanisms in Pickled Chicken Feet: Targeted Inhibition of Spoilage Bacteria

Spoilage of pickled chicken feet is primarily driven by Gram-positive bacteria (G⁺ bacteria), and in some cases, it involves synergistic effects with Gram-negative bacteria (G⁻ bacteria) and fungi. Nisin specifically inhibits the growth of spoilage bacteria through three mechanisms—"disrupting bacterial cell membranes," "inhibiting spore germination," and "interfering with metabolic pathways"—thereby delaying the spoilage process at its source.

(I) Disrupting Gram-Positive Bacterial Cell Membranes and Inducing Cell Lysis

The core antibacterial target of Nisin is the cell membrane of G⁺ bacteria. Special amino acid residues in its molecular structure, such as lanthionine and β-methyl lanthionine, can specifically bind to lipid II (a cell wall precursor containing pyrophosphate groups) on bacterial cell membranes, forming a "Nisin-lipid II complex." This complex further constructs transmembrane channels in the cell membrane:After the formation of transmembrane channels, small molecules inside the bacterial cell—such as potassium ions (K⁺), protons (H⁺), and amino acids—leak out in large quantities, causing osmotic imbalance. At the same time, external water and harmful substances enter the cell, ultimately leading to cell membrane rupture and cell lysis.

For Staphylococcus aureus—the most common spoilage bacterium in pickled chicken feet, which can produce enterotoxins and cause food poisoning—the minimum inhibitory concentration (MIC) of Nisin is only 0.125–0.5 μg/mL, far below its safe upper limit for food addition (GB 2760 National Food Safety Standard for the Use of Food Additives specifies ≤ 0.5 g/kg in meat products). It can rapidly inhibit the growth of this bacterium at low doses: adding 0.2 g/kg Nisin at 25℃ reduces the colony count of Staphylococcus aureus from 10⁵ CFU/g to below 10² CFU/g within 6 hours.

Nisin also exerts a strong inhibitory effect on Listeria monocytogenes—a high-risk bacterium that tends to proliferate at low temperatures (0–4℃) in pickled chicken feet, with cold tolerance and high pathogenicity. By disrupting the integrity of its cell membrane, even under 4℃ refrigeration, adding 0.3 g/kg Nisin can reduce the proliferation rate of this bacterium by more than 80%, avoiding "latent contamination" during low-temperature storage.

(II) Inhibiting Spore Germination and Blocking "Latent Spoilage"

Although high-temperature braising (usually 100–105℃ for 15–20 minutes) during the processing of pickled chicken feet can kill most vegetative bacterial cells, it cannot completely destroy the spores of bacteria such as Bacillus cereus and Bacillus subtilis (spores are heat-resistant and require 121℃ high-pressure sterilization for complete elimination). These spores easily reactivate and germinate into vegetative cells during low-temperature storage, then proliferate in large quantities to cause product spoilage (e.g., producing mucus and releasing spoilage gases)—a phenomenon known as "latent spoilage." Nisin inhibits spore germination through two pathways:

Binding to lipid II on the spore membrane, preventing the synthesis of cell walls required for spore germination and thus blocking the transformation of spores into vegetative cells;

Penetrating into the interior of spores, damaging the DNA and enzyme systems in the spore core, and reducing the reactivation ability of spores.

Experimental data show that adding 0.25 g/kg Nisin during the processing of pickled chicken feet reduces the germination rate of Bacillus cereus spores from 60% to below 15%. After 14 days of refrigeration, the colony count of Bacillus cereus in pickled chicken feet containing Nisin is only 10³ CFU/g, while that in the non-added group reaches 10⁶ CFU/g (exceeding the food hygiene standard limit of 10⁵ CFU/g), effectively blocking latent spoilage.

(III) Synergizing with Acidic Environments to Enhance Antibacterial Spectrum

Pickled chicken feet are acidic foods (pH 3.5–4.5, due to the addition of citric acid, lactic acid, acetic acid, etc.), and the antibacterial activity of Nisin is highly pH-dependent. The acidic environment maintains the structural stability of Nisin (preventing peptide bond hydrolysis) and enhances its permeability to cell membranes. More importantly, acidic conditions can disrupt the outer membrane barrier of G⁻ bacteria (such as Escherichia coli and Salmonella—not the main spoilage bacteria in pickled chicken feet, but potentially introduced through cross-contamination of raw materials). The outer membrane of G⁻ bacteria contains lipopolysaccharides that block the entry of Nisin; acid breaks this barrier, allowing Nisin to act on the cell membrane of G⁻ bacteria and broaden its antibacterial spectrum. For example, in a pickled chicken feet system with pH 4.0, adding 0.3 g/kg Nisin reduces the colony count of Escherichia coli from 10⁴ CFU/g to below 10² CFU/g. In contrast, in a neutral system (pH 7.0), the same dose of Nisin has almost no inhibitory effect on Escherichia coli. This "Nisin + acidic environment" synergistic effect enables Nisin to not only inhibit the main spoilage bacteria in pickled chicken feet but also control potential pathogenic bacteria, improving product safety.

II. Practical Application Effects in Preserving Pickled Chicken Feet: Extending Shelf Life and Ensuring Quality

In the actual production of pickled chicken feet, the antibacterial effects of Nisin are directly reflected in three dimensions: "shelf life extension," "sensory quality maintenance," and "food safety assurance." Its effects are influenced by the addition dose, application method, and storage conditions, and need to be optimized in conjunction with the production process.

(I) Extending Refrigerated Shelf Life and Reducing Spoilage Rate

The conventional refrigerated shelf life (0–4℃) of pickled chicken feet is usually 7–10 days; beyond 10 days, spoilage phenomena such as bag swelling and off-odors are likely to occur. Adding Nisin can significantly slow down the microbial proliferation rate and extend the shelf life to 14–21 days:A study on industrially produced pickled chicken feet showed that in the control group without Nisin, the total colony count reached 5×10⁵ CFU/g on the 10th day of refrigeration (exceeding the limit of 1×10⁵ CFU/g specified in GB 2726 National Food Safety Standard for Cooked Meat Products), with slight off-odors. In contrast, in the experimental group with 0.2 g/kg Nisin, the total colony count was still 8×10⁴ CFU/g on the 14th day of refrigeration, and rose to 1.2×10⁵ CFU/g on the 21st day—with no obvious spoilage signs at this point. If the Nisin dose is increased to 0.3 g/kg, the shelf life can be further extended to 28 days, and the total colony count is always controlled within the safe range.

For pickled chicken feet produced in small workshops (with a higher risk of microbial contamination in the processing environment), the preservation effect of Nisin is more significant: adding 0.3 g/kg Nisin can extend the original shelf life of only 5 days to 12 days, greatly reducing product loss due to spoilage (from 20% to below 5%).

(II) Maintaining Sensory Quality and Avoiding Texture and Flavor Deterioration

The sensory quality of pickled chicken feet (chewy texture, balanced spicy-sour taste) is the core concern of consumers. Microbial proliferation causes quality deterioration by "decomposing proteins" and "producing metabolites": spoilage bacteria secrete proteases that break down collagen and myofibrillar proteins in chicken feet, turning the chewy texture soft; at the same time, bacterial metabolism produces off-odor substances such as ammonia and hydrogen sulfide, destroying the spicy-sour flavor. By inhibiting the growth of spoilage bacteria, Nisin can effectively maintain the sensory quality of pickled chicken feet:

Texture: Pickled chicken feet with 0.25 g/kg Nisin still have a hardness value (a key indicator reflecting chewiness) of 2500–2800 g after 21 days of refrigeration, which is close to that of fresh products (2800–3000 g). In contrast, the hardness value of the non-added group drops to below 1500 g after 10 days of refrigeration, with a significantly soft texture.

Flavor: Nisin itself is odorless and does not react with flavor substances such as capsaicin and citric acid in pickled chicken feet. Pickled chicken feet with Nisin can still maintain a strong spicy-sour taste after 21 days of refrigeration, without spoilage odors such as ammonia or sourness, and their sensory score (out of 10 points) remains above 8 points. In contrast, the sensory score of the non-added group is only 5 points after 14 days of refrigeration (with obvious off-odors).

(III) Reducing Pathogen Risks and Ensuring Food Safety

During the processing of pickled chicken feet, if raw chicken feet are contaminated with bacteria (e.g., Staphylococcus aureus from slaughter) or processing equipment is not thoroughly cleaned (with residual Listeria monocytogenes), pathogenic contamination may occur, leading to food poisoning. Nisin can reduce safety risks by inhibiting the growth of these pathogenic bacteria:

Against Staphylococcus aureus: Adding 0.2 g/kg Nisin to pickled chicken feet can completely inhibit the growth of this bacterium under 4℃ refrigeration. Even if the initial contamination level of raw materials reaches 10⁴ CFU/g, no Staphylococcus aureus is detected after 14 days of refrigeration. In contrast, in the non-added group, the content of Staphylococcus aureus rises to 10⁶ CFU/g after 7 days of refrigeration, and enterotoxins (which may cause vomiting and diarrhea) are detected.

Against Listeria monocytogenes: This bacterium can still proliferate slowly at 0–4℃, and conventional refrigeration cannot control it. After adding 0.3 g/kg Nisin, the proliferation rate of this bacterium in pickled chicken feet can be reduced by 90%, and it never exceeds the safety limit (GB 2726 specifies that Listeria monocytogenes shall not be detected in cooked meat products) within 28 days of refrigeration, effectively avoiding pathogenic risks during low-temperature storage.

III. Key Factors Influencing Nisin’s Antibacterial Effects in Pickled Chicken Feet

The antibacterial effect of Nisin in pickled chicken feet is not constant; it is affected by four key factors: "system pH," "salt and seasonings," "processing technology," and "storage conditions." Improper control of these factors may lead to reduced or even lost antibacterial effects.

(I) System pH: Maintaining an Acidic Range and Avoiding Excessively High pH

As mentioned earlier, Nisin is stable in activity in an acidic environment, and the pH of pickled chicken feet is usually 3.5–4.5, which exactly matches its optimal activity range. However, improper pH control during production (e.g., insufficient addition of acetic acid leading to pH > 5.0) will significantly reduce the antibacterial effect of Nisin:When the pH of pickled chicken feet rises to 5.5, the molecular structure of Nisin is prone to conformational changes, and the hydrolysis rate of peptide bonds accelerates, resulting in a 40% loss of activity within 24 hours. At this point, the MIC of Nisin against Staphylococcus aureus increases from 0.25 μg/mL to 1.0 μg/mL, and double the amount of Nisin needs to be added to achieve the original antibacterial effect.

In practice, the final pH of pickled chicken feet must be strictly controlled (monitored in real time with a pH meter to ensure it is within 3.8–4.2) to avoid pH fluctuations caused by improper seasoning. If flavor adjustment is needed (e.g., reducing acidity), sodium citrate (a buffer) can be added to maintain pH stability instead of reducing the amount of acidic seasonings.

(II) Salt and Seasonings: High Salt and Some Spices May Weaken the Effect

Pickled chicken feet are high-salt foods (salt addition is usually 2%–3%) and contain spices such as chili, Sichuan pepper, and star anise. These components may interact with Nisin, affecting its antibacterial activity:

High salt: A NaCl concentration > 3% weakens the binding ability of Nisin to bacterial cell membranes through the "charge shielding effect"—Cl⁻ ions dissociated from NaCl bind to positively charged groups in the Nisin molecule, reducing the affinity of Nisin for lipid II and leading to decreased antibacterial activity (e.g., at a 3% salt concentration, the inhibition rate of Nisin against Bacillus cereus drops from 90% to 60%).

Some spices: Spices such as star anise and cinnamon contain polyphenols, which form hydrogen-bonded complexes with Nisin, causing it to lose free activity. Experiments show that in pickled chicken feet with 1% star anise powder added, the effective concentration of Nisin decreases by 30%, and an additional 0.1 g/kg Nisin needs to be supplemented to maintain the antibacterial effect.

Countermeasures: Control the amount of salt added within 2%–2.5% to avoid excessive levels; select "low-polyphenol spices" (e.g., chili and Sichuan pepper have low polyphenol content and can be added normally; the amount of star anise and cinnamon should be controlled below 0.3%); or let the mixture stand for 30 minutes after adding spices before adding Nisin to reduce direct interaction between polyphenols and Nisin.

(III) Processing Technology: Avoiding High-Temperature, Long-Duration Treatment to Prevent Nisin Inactivation

The processing of pickled chicken feet usually includes the steps of "braising (100℃ for 15 minutes) → cooling → flavoring and marinating → packaging." The high temperature during the braising step may inactivate Nisin, so attention must be paid to the timing of addition:If Nisin is added before braising, heating at 100℃ for 15 minutes will cause more than 90% of its activity to be lost (high temperature destroys its lanthionine structure), and it will almost lose its antibacterial effect. In contrast, adding Nisin after braising and cooling (when the temperature of chicken feet drops below 40℃) can control the activity loss within 10%, allowing Nisin to fully exert its antibacterial effect.

In addition, the marinating time also needs to be controlled (usually 4–6 hours at 25℃). If the marinating time is too long (> 12 hours), Nisin may be degraded by endogenous proteases in chicken feet (e.g., cathepsins), and its activity will gradually decrease (about 25% loss of activity after 12 hours of marinating). In practice, the marinating time must be strictly controlled to ensure that packaging is completed within 6 hours.

(IV) Storage Conditions: Low-Temperature Refrigeration Is Key to Avoiding Temperature Fluctuations

Although Nisin can inhibit microbial growth, it cannot completely kill existing bacteria. Low-temperature storage (0–4℃) is required to maximize the shelf life. If the storage temperature fluctuates (e.g., cold chain breakage causes the temperature to rise above 10℃), microbial proliferation will be accelerated, offsetting the antibacterial effect of Nisin:At 4℃ refrigeration, the shelf life of pickled chicken feet with 0.2 g/kg Nisin is 21 days; if the temperature rises to 10℃, the microbial proliferation rate will accelerate by 3–4 times, and the shelf life will be shortened to 7 days, which is close to that of products without Nisin added.

At the same time, repeated thawing (e.g., consumers take pickled chicken feet out of the refrigerator to rewarm and then put them back for refrigeration) will cause Nisin to be lost with moisture during thawing, reducing its effective concentration; moreover, thawing will damage the cell membrane structure of chicken feet, providing more nutrients for microorganisms and accelerating spoilage.

Countermeasures: Maintain cold chain storage throughout the entire process (control the temperature at 0–4℃ from post-production cooling, transportation, and sales to consumer storage); use "small independent packaging" to avoid repeated thawing by consumers, reducing the risk of Nisin loss and microbial contamination.

IV. Optimized Application Strategies for Nisin in Pickled Chicken Feet Preservation

To maximize the antibacterial effect of Nisin, optimized strategies must be developed based on the aforementioned influencing factors, focusing on three dimensions: "synergistic enhancement via compounding," "precision addition," and "process adaptation." These strategies aim to achieve the goals of "low dosage, high efficiency, and long shelf life."

(I) Compounding with Other Antibacterial Agents to Expand Antibacterial Spectrum and Enhance Efficacy

Nisin has weak inhibitory effects on Gram-negative (G⁻) bacteria and reduced activity in high-salt environments. Compounding it with natural antibacterial agents (such as chitosan and ε-polylysine) achieves synergistic enhancement:

Nisin + Chitosan (compound ratio 1:10, 0.2 g/kg Nisin + 2.0 g/kg chitosan):Chitosan can disrupt the outer membrane of G⁻ bacteria, allowing Nisin to act on G⁻ bacteria. Additionally, chitosan’s film-forming property creates a protective layer on the surface of chicken feet, slowing down Nisin loss. This compounding scheme extends the shelf life of pickled chicken feet from 21 days to 28 days and inhibits G⁻ bacteria such as Escherichia coli and Salmonella.

Nisin + ε-Polylysine (compound ratio 1:5, 0.2 g/kg Nisin + 1.0 g/kg ε-polylysine):ε-Polylysine inhibits both Gram-positive (G⁺) and G⁻ bacteria. When combined with Nisin, it enhances the inhibitory effect on microorganisms in high-salt environments (e.g., at a 3% salt concentration, the inhibition rate increases from 60% to 85%). Simultaneously, it reduces the required Nisin dosage (from 0.3 g/kg to 0.2 g/kg), lowering production costs.

(II) Precision Control of Addition Timing and Method to Ensure Uniform Distribution

The timing and method of Nisin addition directly affect its uniform distribution in pickled chicken feet and the retention of its activity:

Addition Timing:Nisin must be added strictly after braising and cooling (when chicken feet temperature < 40℃) and before flavoring and marination to avoid inactivation by high temperatures and degradation by proteases. If a "vacuum marination" process is adopted (vacuum degree -0.08 MPa, 25℃, 4 hours), Nisin can be added to the marinating solution. Vacuum infiltration ensures Nisin is uniformly distributed inside the chicken feet (not just on the surface), improving the inhibitory effect on potential internal bacteria.

Addition Method:First, dissolve Nisin in a small amount of sterile water (or the braising broth of pickled chicken feet) to prepare a 10 g/L Nisin solution. Then, spray the solution evenly or add it to the marinating solution. Direct addition of Nisin powder should be avoided—the powder tends to agglomerate, resulting in uneven distribution (local high concentrations and insufficient concentrations in other areas), which undermines uniform antibacterial efficacy.

(III) Adapting to Processing Technology to Reduce Activity Loss

Optimize process parameters based on the pickled chicken feet production workflow to minimize Nisin activity loss:

Braising Process:Control the braising temperature at 95–100℃ and duration at 12–15 minutes (instead of 105℃ for 20 minutes). This ensures chicken feet are fully cooked while shortening subsequent cooling time, preventing early microbial growth. It is required to cool chicken feet from 100℃ to below 40℃ within 2 hours; rapid cooling via an ice-water bath is recommended.

Packaging Process:Adopt a combined "vacuum packaging + pasteurization" process (after vacuum packaging, perform pasteurization at 60℃ for 30 minutes). Pasteurization kills most residual vegetative bacterial cells, reducing the antibacterial burden on Nisin. Meanwhile, the vacuum environment inhibits the growth of aerobic bacteria, which synergizes with Nisin to extend shelf life (extending it by an additional 7 days compared to vacuum packaging alone).

Nisin exhibits significant effects in pickled chicken feet preservation, including "targeted inhibition of spoilage bacteria, shelf life extension, quality maintenance, and safety assurance." Its core mechanisms involve disrupting G⁺ bacterial cell membranes, inhibiting spore germination, and synergizing with the acidic environment of pickled chicken feet to expand the antibacterial spectrum.

In practical applications, to maximize Nisin’s antibacterial efficacy, strict control of the system pH (3.8–4.2), salt content (2%–2.5%), and "post-cooling addition" timing is essential, combined with 0–4℃ refrigeration. Further efficacy improvements can be achieved by compounding with chitosan or ε-polylysine, or by adapting processes such as "vacuum marination + pasteurization"—extending the refrigerated shelf life of pickled chicken feet from 7–10 days to 21–28 days.

It should be noted that Nisin is an "auxiliary preservation tool" and cannot replace good manufacturing practices (such as raw material cleaning and equipment disinfection). The food safety of pickled chicken feet must still be centered on "source control + process optimization + cold chain management." Nisin’s value lies in improving preservation efficiency and reducing spoilage losses.

In the future, with in-depth research on the interaction mechanisms between Nisin and food matrices, its application in pickled chicken feet and other low-temperature meat products will become more precise, providing safer and more efficient solutions for food preservation technology.