The inhibitory effect of Nisin in combination with organic acids

The combination of Nisin and organic acids (e.g., citric acid, lactic acid, acetic acid, malic acid) produces a significant synergistic inhibitory effect. It not only enhances Nisin’s inhibitory effect on Gram-positive bacteria but also overcomes its limitations in inhibiting Gram-negative bacteria and fungi. Meanwhile, it reduces the dosage of both when used alone, aligning with the "low addition, high safety" requirements of green food, and is widely applied in meat products, dairy products, fruit and vegetable products, and other fields.

I. Synergistic Inhibition Mechanism of the Combination: From "Single Action" to "Multi-Target Enhancement"

The synergistic inhibition of Nisin and organic acids primarily relies on three mechanisms—"destroying bacterial barriers, enhancing Nisin permeability, and altering microbial metabolic environments"—to amplify the antibacterial effect. The specific principles are as follows:

(I) Organic Acids Destroy Bacterial Cell Membranes and Enhance Nisin Permeability

Nisin’s core antibacterial target is the cell membrane of Gram-positive bacteria. However, Gram-negative bacteria have a "lipopolysaccharide layer (LPS)" outside their cell walls, which blocks Nisin penetration, resulting in weak inhibitory effects of Nisin on Gram-negative bacteria. Organic acids can destroy this barrier in two ways:

Lowering extracellular pH: Organic acids (e.g., citric acid, pKa ≈ 3.13) release H⁺ in aqueous solutions, reducing the environmental pH below the optimal growth range of bacteria (most pathogenic bacteria thrive at pH 6.0–7.5). This disrupts the bacterial cell membrane potential and loosens the structure of the lipopolysaccharide layer.

Direct action of acid molecules: Undissociated organic acid molecules can penetrate the bacterial cell membrane. After entering the cell, they dissociate to release H⁺, lowering the intracellular pH, inhibiting enzyme activity (e.g., glycolytic enzymes, tricarboxylic acid cycle enzymes), and damaging the integrity of the cell membrane. For example, acetic acid can dissolve the lipopolysaccharide layer of Gram-negative bacteria, forming "channels" in the cell membrane that allow Nisin to enter the cell and act on membrane targets.

This synergistic effect significantly broadens Nisin’s antibacterial spectrum: Using 0.05 g/kg Nisin alone has almost no inhibitory effect on E. coli (a Gram-negative bacterium), but when combined with 0.2% citric acid, the inhibition rate of E. coli can reach over 92%, and the dosage of Nisin can be reduced by 30%.

(II) Organic Acids Enhance Nisin Stability and Extend Antibacterial Duration

Nisin is prone to degradation in neutral or alkaline environments (e.g., at pH > 7, its activity decreases by 50% within 24 hours) and is easily decomposed by proteases produced by bacteria (e.g., lactic acid bacteria proteases). Organic acids can improve its stability in two aspects:

Maintaining an acidic environment: Organic acids stabilize the system pH at 3.5–5.0 (the optimal pH range for Nisin activity), slowing down its chemical degradation rate. For example, in yogurt at pH 4.0, the activity retention rate of Nisin is 40% higher than that in milk at pH 6.5.

Inhibiting protease activity: Most bacterial proteases (e.g., subtilisin) have reduced activity in acidic environments. Organic acids can reduce the decomposition of Nisin by proteases. For example, in meat products, combining Nisin with lactic acid extends Nisin’s half-life from 10 days to 18 days, significantly prolonging its antibacterial duration.

(III) Altering Microbial Metabolism and Inhibiting Drug Resistance Development

Long-term single use of Nisin may lead to bacterial drug resistance (e.g., some lactic acid bacteria resist Nisin by synthesizing "Nisin resistance proteins"). Organic acids can reduce the risk of drug resistance by altering microbial metabolic pathways:

Organic acids inhibit bacterial "energy metabolism," preventing bacteria from synthesizing drug resistance-related proteins (e.g., transporters, modification enzymes).

The combination acts on different targets of microorganisms (Nisin destroys cell membranes, while organic acids inhibit enzyme activity). Bacteria rarely develop resistance to both mechanisms simultaneously, thus maintaining long-term antibacterial effects.

II. Differences in Inhibitory Effects of Nisin Combined with Different Organic Acids

Different organic acids have varying pKa values and molecular structures, leading to differences in their inhibitory effects when combined with Nisin. Suitable organic acids should be selected based on food categories (acidic/neutral, liquid/solid). The specific differences are as follows:

(I) Citric Acid (pKa ≈ 3.13): Broad-Spectrum Synergy, Suitable for Most Foods

Citric acid is the most widely used organic acid. Its combination with Nisin features "broad-spectrum antibacterial activity and mild flavor":

Antibacterial advantages: It has a synergistic inhibitory effect on Gram-positive bacteria (e.g., Listeria), Gram-negative bacteria (e.g., Salmonella), and fungi (e.g., yeast). It is particularly suitable for acidic foods (e.g., fruit juice, yogurt) and neutral foods (e.g., meat products, canned foods).

Application case: In low-temperature ham, combining 0.03 g/kg Nisin with 0.3% citric acid results in a Listeria count of < 10 CFU/g, extending the shelf life from 15 days to 30 days. Additionally, the sour taste of citric acid masks the slight bitterness of Nisin, improving the ham’s flavor.

(II) Lactic Acid (pKa ≈ 3.86): High Permeability, Suitable for Meat and Dairy Products

Lactic acid has a small molecular size and high permeability. Its combination with Nisin works exceptionally well in high-protein foods:

Antibacterial advantages: Lactic acid can penetrate the muscle fiber gaps of meat, acting with Nisin on deep-seated bacteria (preventing "internal contamination" of meat products). In dairy products, it promotes Nisin dissolution and reduces precipitation.

Application case: In cheese production, combining 0.02 g/kg Nisin with 0.2% lactic acid inhibits miscellaneous bacteria (e.g., acid-producing bacteria) during cheese ripening, preventing excessive sourness and extending the shelf life from 60 days to 90 days.

(III) Acetic Acid (pKa ≈ 4.76): Strong Bactericidal Activity, Suitable for High-Salt and Fermented Foods

Acetic acid has strong volatility and bactericidal activity. Its combination with Nisin is suitable for high-salt or fermented scenarios:

Antibacterial advantages: Acetic acid has a significant inhibitory effect on salt-tolerant bacteria (e.g., Vibrio parahaemolyticus). In fermented foods (e.g., pickles), it works synergistically with Nisin to inhibit excessive fermentation of lactic acid bacteria.

Application case: In pickles, combining 0.015 g/kg Nisin with 0.4% acetic acid reduces the nitrite content of pickles by 50%, inhibits yeast growth to prevent "bag swelling," and extends the shelf life from 20 days to 40 days.

(IV) Malic Acid (pKa ≈ 3.40): Flavor Compatibility, Suitable for Fruit/Vegetable Products and Beverages

Malic acid has a natural fruity flavor. Its combination with Nisin balances antibacterial effects and flavor in fruit/vegetable products and beverages:

Antibacterial advantages: Malic acid has a mild sour taste that improves the texture of fruit/vegetable products. It also works synergistically with Nisin to inhibit Pectobacterium (preventing soft rot) and Penicillium (preventing mold) in fruits and vegetables.

Application case: In fresh-cut apples, soaking them in a combination of 0.02 g/kg Nisin and 0.2% malic acid and storing at 4°C reduces the browning rate of apples by 60%, extends the period for meeting the total bacterial count standard from 3 days to 7 days, and retains the natural flavor of apples.

III. Key Control Factors for Combined Application

The inhibitory effect of the Nisin-organic acid combination is significantly affected by the combination ratio, system pH, and food matrix. Targeted control is required to maximize the synergistic effect:

(I) Combination Ratio: Avoid "Excessive Dosage" or "Insufficient Synergy"

High-risk foods (e.g., low-temperature meat products, sterile dairy products): Nisin dosage is 0.03–0.05 g/kg, and organic acid dosage is 0.2%–0.4% (e.g., 0.04 g/kg Nisin + 0.3% citric acid) to ensure antibacterial strength.

Low-risk foods (e.g., fresh-cut fruits/vegetables, baked goods): Nisin dosage is 0.01–0.02 g/kg, and organic acid dosage is 0.1%–0.2% (e.g., 0.015 g/kg Nisin + 0.15% malic acid) to avoid excessive sourness caused by overuse of organic acids.

Precautions: Organic acid dosage should not exceed 0.5%, otherwise it may cause sour taste in food (e.g., overly sour meat products) or damage nutrients (e.g., high-concentration acids decompose vitamin C).

(II) System pH: Maintain the Optimal Activity Range of Nisin

Nisin has the strongest activity at pH 3.5–5.0, so organic acids need to control the system pH within this range:

Neutral foods (e.g., meat products, milk): Sufficient organic acids (e.g., 0.3%–0.4% citric acid) should be added to lower the pH to 4.5–5.0, preventing Nisin degradation.

Acidic foods (e.g., fruit juice, yogurt, pH 3.0–4.0): No additional pH adjustment is needed; Nisin only needs to be added in proportion, and organic acids can directly exert a synergistic effect.

(III) Food Matrix: Adapt to Protein and Fat Content

High-protein foods (e.g., meat products, cheese): Lactic acid (with high permeability) is preferred to avoid excessive binding of citric acid to proteins (which causes protein precipitation).

High-fat foods (e.g., cooking oil, fried foods): Malic acid (with good compatibility with fats) is preferred to avoid acetic acid (with high volatility) causing fat oxidation.

High-sugar foods (e.g., jam, fruit juice beverages): The dosage of organic acids can be appropriately increased (e.g., by 20%) to offset the "protective effect" of high sugar on microorganisms (high-sugar environments may reduce the permeability of antibacterial agents).

The inhibitory effect of the Nisin-organic acid combination essentially achieves synergistic enhancement through "destroying bacterial barriers, enhancing Nisin stability, and inhibiting drug resistance." It not only broadens the antibacterial spectrum and extends the shelf life but also reduces the dosage of both components, aligning with green food requirements. The characteristics of different organic acids determine their suitable scenarios (e.g., citric acid for broad-spectrum use, lactic acid for high-protein foods, malic acid for fruit/vegetable products). In practical applications, the combination ratio and pH should be adjusted based on food categories and matrix characteristics to ensure antibacterial effects while balancing flavor and nutrition. In the future, with the refinement of combination technologies (e.g., microencapsulation to improve stability), the application of this system in high-end foods and organic foods will be further expanded.