The application of Nisin in beverages and its stability

As a natural antimicrobial peptide, Nisin has emerged as a crucial alternative to chemical preservatives in the beverage industry, attributed to its high-efficiency inhibition of Gram-positive bacteria, food-grade safety, and partial processing stability. Beverages are susceptible to contamination by bacteria (e.g., lactococci, streptococci, bacilli) during production and storage, leading to rancidity, turbidity, and flavor deterioration. By targeting bacterial cell membrane disruption and cell wall synthesis inhibition, Nisin effectively delays the spoilage process while aligning with consumers’ demand for "natural and additive-free" foods. This article systematically analyzes Nisin’s application scenarios in beverages, factors influencing its stability, synergistic enhancement strategies, and future development trends, providing theoretical and practical references for preservation in the beverage industry.

I. Applications in Different Types of Beverages

Nisin’s antimicrobial spectrum is dominated by Gram-positive bacteria, with significant inhibitory effects on lactic acid bacteria, streptococci, and bacilli that cause beverage rancidity. Its application scenarios require differentiated adaptation based on beverage composition, pH value, and processing technology:

1. Dairy Beverages (Milk-containing Beverages, Fermented Dairy Beverages)

Dairy beverages are rich in protein and lactose, making them prone to contamination by lactococci, Streptococcus thermophilus, and Bacillus cereus, resulting in increased acidity, stratification, and off-odors. As a milk-derived natural preservative, Nisin exhibits excellent compatibility with product characteristics:

Adding 0.05~0.1 g/kg Nisin to milk-containing beverages effectively inhibits Gram-positive bacterial growth. Under room temperature storage (25℃), the total bacterial count is reduced by 2~3 log cycles compared to the control group, extending the shelf life from 7~10 days to 15~21 days.

In fermented dairy beverages (e.g., yogurt drinks), Nisin can be added in the late fermentation stage (to avoid affecting the activity of fermentative strains), inhibiting miscellaneous bacterial contamination, delaying post-acidification, and maintaining flavor stability. The pH decrease during storage is reduced from 0.5~0.8 to 0.2~0.3.

For low-fat/fat-free dairy beverages, the reduced fat content weakens the antimicrobial barrier. The Nisin dosage can be increased to 0.1~0.15 g/kg, or combined with EDTA (0.05%~0.1%) to expand the antimicrobial spectrum to include some Gram-negative bacteria.

2. Plant Protein Beverages (Soy Milk, Walnut Milk, Almond Milk)

Plant protein beverages are centered on plant proteins, making them susceptible to contamination by bacilli (e.g., Bacillus subtilis) and molds. High-temperature sterilization during processing is often ineffective in completely eliminating spores, and Nisin’s spore-inhibiting properties specifically address this issue:

Adding 0.08~0.12 g/kg Nisin to soy milk combined with UHT sterilization (135℃, 5~8 seconds) significantly reduces spore survival rate. After 6 months of room temperature storage, the spore count is reduced by over 90% compared to the control group, with no turbidity or precipitation.

For nut beverages such as walnut milk and almond milk, which contain a certain amount of oil prone to oxidative rancidity, combining Nisin with natural antioxidants (e.g., vitamin E, tea polyphenols) inhibits both bacterial growth and oil oxidation. The peroxide value (POV) is reduced by 30%~40% compared to the control group, extending the shelf life to over 12 months.

Low-sugar plant protein beverages have enhanced microbial tolerance due to reduced sugar content, requiring a composite scheme of "Nisin + calcium propionate (0.05%~0.1%)" to improve preservation efficacy.

3. Fruit Juice Beverages (Concentrated Fruit Juice, Freshly Squeezed Fruit Juice)

Fruit juice beverages have a low pH value (3.0~4.5), and the acidic environment enhances Nisin’s antimicrobial activity. However, components such as vitamin C and polyphenols in some juices may affect its stability:

Adding 0.06~0.1 g/kg Nisin to concentrated fruit juice inhibits the growth of acid-tolerant bacteria such as Lactobacillus acidophilus and streptococci. After 12 months of room temperature storage, there are no signs of rancidity, effectively preserving the juice’s flavor and nutritional components.

Freshly squeezed fruit juice (unsterilized) is highly susceptible to microbial contamination with an extremely short shelf life. Adding 0.05~0.08 g/kg Nisin extends the shelf life from 2~3 days to 7~10 days under refrigerated storage (4℃) without affecting the juice’s color and taste.

In high-sugar fruit juice beverages (e.g., lychee juice, mango juice), the high sugar concentration (>15%) synergizes with Nisin to inhibit microbial growth. The dosage can be reduced to 0.04~0.06 g/kg to avoid the slight bitterness caused by excessive addition.

4. Functional Beverages (Sports Drinks, Energy Drinks)

Functional beverages typically contain electrolytes, vitamins, caffeine, and other components, with a pH value of 3.5~4.5, making them prone to bacterial contamination and flavor deterioration. Nisin synergizes with functional components to improve product stability:

Adding 0.05~0.08 g/kg Nisin to sports drinks inhibits the growth of lactic acid bacteria and bacilli. After 6 months of room temperature storage, the total bacterial count still meets food safety standards (≤10² CFU/mL), and the electrolyte (sodium, potassium) content remains stable.

In caffeine-containing energy drinks, caffeine’s weak antimicrobial activity synergizes with Nisin to further improve preservation efficacy. Adding 0.04~0.06 g/kg Nisin achieves long-term preservation without affecting caffeine’s physiological activity.

Low-sugar/sugar-free functional beverages (using artificial sweeteners) have no microbial inhibitory effect from sweeteners. The Nisin dosage needs to be increased to 0.08~0.1 g/kg, or combined with sodium citrate (0.1%~0.2%) to enhance antimicrobial effects.

5. Other Beverages (Tea Beverages, Carbonated Beverages)

Tea beverages are rich in tea polyphenols, which inherently possess certain antimicrobial activity. Combined use with Nisin produces a synergistic effect. Adding 0.03~0.05 g/kg Nisin inhibits bacterial growth, with no turbidity or off-odors after 6 months of room temperature storage, and the tea polyphenol retention rate is increased by 10%~15%.

Carbonated beverages have a low microbial contamination risk due to the antimicrobial effect of CO₂. Only fruit juice-type carbonated beverages require the addition of 0.02~0.04 g/kg Nisin to inhibit miscellaneous bacterial contamination from fruit juice, extending the shelf life to over 12 months.

II. Factors Influencing Nisin Stability in Beverages

The activity stability of Nisin in beverages directly determines its preservation efficacy, regulated by multiple factors such as beverage composition, pH value, processing technology, and storage conditions. The core influencing mechanisms are as follows:

1. Influence of pH Value

pH value is a key factor affecting Nisin activity, with optimal stability and antimicrobial activity in acidic environments:

At pH 3.0~5.0, the Nisin molecular structure is stable, and antimicrobial activity is strongest (minimum inhibitory concentration MIC reduced by 30%~50%), which highly aligns with the pH range of most beverages (fruit juices, dairy beverages, functional beverages).

At pH > 6.0 (e.g., some neutral plant protein beverages, milk-containing beverages), Nisin molecules are prone to conformational changes, resulting in significant activity loss. At pH 7.0, activity loss can reach over 50%, and at pH 8.0, it is almost completely inactivated.

At pH < 2.5 (e.g., high-acidity fruit juices), Nisin retains activity but may undergo slow acid-catalyzed hydrolysis during long-term storage, leading to gradual activity decline. Stabilizers (e.g., xanthan gum, pectin) need to be added to reduce hydrolysis.

2. Influence of Processing Technology

Heat treatment, homogenization, sterilization, and other steps in beverage processing affect Nisin activity, requiring targeted optimization of process parameters:

Heat Treatment: Nisin exhibits good stability under low-temperature short-time heat treatment (e.g., pasteurization, 65℃ for 30 minutes) with activity loss < 10%; under high-temperature sterilization (e.g., UHT, 135℃ for 5 seconds), activity loss is approximately 20%~30%; ultra-high temperature long-time sterilization (>140℃ for >10 seconds) results in activity loss of over 50%. Therefore, Nisin should be added in the cooling stage after sterilization, or the dosage appropriately increased to compensate for losses.

Homogenization: High-pressure homogenization (20~30 MPa) has minimal impact on Nisin activity (loss < 5%) and promotes uniform dispersion in beverages, enhancing antimicrobial efficacy.

Sterilization Methods: Non-thermal sterilization technologies such as microwave sterilization and pulsed electric field sterilization have far less impact on Nisin activity than traditional thermal sterilization (activity loss < 10%), making them more suitable for combined use with Nisin to achieve the synergistic effect of "low-temperature sterilization + natural preservation."

3. Influence of Beverage Components

Components such as proteins, fats, sugars, and additives in beverages may interact with Nisin, affecting its free concentration and activity:

Proteins and Fats: Proteins (e.g., casein, soy protein) and fats in dairy beverages and plant protein beverages bind to Nisin through hydrophobic interactions and hydrogen bonds, reducing free Nisin concentration and antimicrobial activity. The dosage needs to be appropriately increased (20%~30% higher than in protein-free beverages).

Sugars and Artificial Sweeteners: Sucrose, fructose, and other sugars synergize with Nisin to inhibit microbial growth by reducing water activity (Aw), but high-concentration sugars (>20%) may encapsulate Nisin molecules, slightly reducing activity; artificial sweeteners such as aspartame and stevioside have no significant impact on Nisin activity.

Additives: Chelating agents such as EDTA and citric acid enhance Nisin’s inhibitory effect on Gram-negative bacteria and improve stability when used in combination; reducing agents such as sulfites may destroy disulfide bonds in Nisin molecules, leading to activity loss and should be avoided.

4. Influence of Storage Conditions

The activity decay rate of Nisin during beverage storage is closely related to temperature, light, and oxygen content:

Temperature: Refrigerated storage (0~4℃) significantly delays Nisin activity decay, with a retention rate of over 80% after 6 months; room temperature storage (25℃) results in a retention rate of approximately 60%~70%; high-temperature storage (>30℃) accelerates hydrolysis, with activity loss of over 50% after 3 months.

Light: Ultraviolet light damages the Nisin molecular structure, leading to activity decline. Beverages should use light-proof packaging (e.g., brown bottles, aluminum foil packaging) to reduce light exposure.

Oxygen Content: Oxygen accelerates Nisin oxidation and inactivation, particularly in acidic beverages. Vacuum packaging or modified atmosphere packaging (filled with N₂/CO₂ mixed gas) extends the active shelf life of Nisin.

III. Optimization Strategies to Enhance Nisin Stability and Antimicrobial Efficacy in Beverages

To address issues such as low Nisin activity in neutral beverages, susceptibility to component interference, and limited antimicrobial spectrum, synergistic enhancement can be achieved through composite formulations, process optimization, and formulation modification:

1. Composite Preservation Schemes

Combining Nisin with other preservatives or functional components achieves "synergistic enhancement and expanded antimicrobial spectrum" while reducing the dosage of individual components:

Combination with Chelating Agents: Nisin + EDTA (0.05%~0.1%) is the most commonly used composite scheme in beverages. EDTA chelates calcium ions on the cell membrane of Gram-negative bacteria, disrupting membrane integrity and facilitating Nisin entry into cells. This inhibits Gram-negative bacteria such as Escherichia coli and Salmonella, suitable for neutral plant protein beverages and milk-containing beverages.

Combination with Natural Preservatives: Nisin + plant extracts (e.g., clove essential oil, cinnamon essential oil, dosage 0.01%~0.05%) or Nisin + probiotic metabolites leverages the antimicrobial and antioxidant properties of natural ingredients, enhancing the "natural" attribute of products while improving preservation efficacy, suitable for fruit juice beverages and tea beverages.

Combination with Chemical Preservatives: Nisin + potassium sorbate (0.05%~0.1%) or Nisin + sodium dehydroacetate (0.03%~0.05%) inhibits both bacteria and molds, suitable for low-sugar and additive-free beverages. The dosage can be reduced by 40%~50% compared to single use, minimizing chemical preservative residues.

2. Process Optimization and Addition Method Adjustment

Addition Timing: Nisin is preferably added in the cooling stage after sterilization (temperature < 50℃) to avoid activity loss from heat treatment; for fermented beverages (e.g., yogurt drinks), it should be added after fermentation and before refrigeration to avoid affecting fermentative strains.

Addition Method: Dissolve Nisin in a small amount of sterile water or acidic solution (e.g., citric acid solution), stir uniformly, and add to beverages to ensure uniform dispersion; for beverages high in protein and fat, Nisin can be mixed with emulsifiers (e.g., glycerol monostearate) before addition to reduce binding with proteins and fats.

Process Parameter Optimization: Adopt a composite process of "low-temperature sterilization + Nisin," such as reducing UHT sterilization temperature from 135℃ to 125℃ and shortening time to 3~5 seconds, reducing Nisin activity loss while ensuring sterilization efficacy; for neutral beverages, appropriately adjust pH to 5.0~5.5 (by adding a small amount of citric acid) to enhance Nisin activity.

3. Formulation Modification Technology

Modifying Nisin through formulation technology enhances its stability, dispersion, and targeting:

Microencapsulation: Encapsulating Nisin with wall materials such as gum arabic, maltodextrin, and β-cyclodextrin protects it from beverage components (proteins, fats) and processing technology, reducing activity loss, achieving controlled release, and extending preservation duration, suitable for neutral beverages and high-sugar beverages.

Molecular Modification: Altering Nisin’s charge characteristics and conformational stability through molecular modification technologies such as acetylation and PEGylation enhances its activity and hydrolysis resistance in neutral environments, expanding application scenarios.

Nanocomposite Formulations: Loading Nisin onto nanocarriers (e.g., nanocellulose, liposomes) improves dispersion uniformity in beverages, enhances binding capacity with bacterial cell membranes, improves antimicrobial efficiency, and reduces dosage.

4. Product Formulation Adaptation Adjustment

For neutral beverages (pH > 6.0), add a small amount of organic acids such as citric acid and malic acid to adjust pH to 5.0~5.5, enhancing Nisin activity; or increase EDTA dosage to 0.1%~0.15% to compensate for activity loss in neutral environments.

For high-protein and high-fat beverages, appropriately reduce protein and fat content (e.g., low-fat dairy beverages) or increase emulsifier dosage to reduce Nisin binding with proteins and fats, improving free Nisin concentration.

Optimize beverage water activity (Aw) by adding sugars or polyols (e.g., glycerol, sorbitol) to control Aw below 0.85, synergizing with Nisin to inhibit microbial growth and improve stability.

IV. Challenges and Future Development Trends

1. Existing Challenges

Limited Antimicrobial Spectrum: Nisin has weak direct inhibitory effects on Gram-negative bacteria and molds, relying on composite schemes, which increases application complexity.

Poor Applicability in Neutral Beverages: Significant activity decline in neutral beverages with pH > 6.0 limits its application in some plant protein beverages and sports drinks.

Sensory Impact: High-concentration addition (>0.15 g/kg) may cause slight bitterness and off-odors, affecting beverage taste.

High Cost: Fermentation and purification costs of Nisin are higher than traditional chemical preservatives, limiting large-scale application in mid-to-low-end beverages.

2. Development Trends

Development of Modified Nisin: Through genetic engineering modification of fermentation strains or molecular modification technologies, develop Nisin derivatives suitable for neutral environments with a broader antimicrobial spectrum, enhancing activity and stability in neutral beverages.

Low-Cost Production Processes: Optimize Nisin fermentation strains (e.g., breeding high-yield strains), culture media (e.g., using agricultural waste as carbon sources), and purification processes to reduce production costs and expand application scope.

Multifunctional Composite Formulations: Develop composite formulations integrating preservation, oxidation resistance, and nutritional fortification, such as Nisin + vitamin C + plant extract composite formulations, simplifying beverage formulations and improving product added value.

Synergy with Intelligent Packaging: Load Nisin onto intelligent packaging materials (e.g., biodegradable films, sustained-release coatings) to achieve controlled release during storage, extending preservation duration, avoiding interactions between Nisin and beverage components, and improving stability.

Personalized Application Schemes: Develop customized Nisin composite preservation schemes for different beverage types based on their composition characteristics and processing technologies, such as the "Nisin + EDTA + microencapsulation" scheme for neutral plant protein beverages and the "low-dose Nisin + tea polyphenols" scheme for high-acidity fruit juices.

As a natural antimicrobial peptide, Nisin has broad application prospects in the beverage industry. It effectively inhibits the proliferation of Gram-positive bacteria and spores, delays product rancidity, turbidity, and flavor deterioration, while aligning with the "natural and healthy" consumption trend. Its application efficacy and stability are influenced by factors such as pH value, processing technology, and beverage composition. Through composite preservation schemes, process optimization, and formulation modification, its stability and antimicrobial efficiency can be significantly enhanced to meet the needs of different beverage types.

Despite current challenges such as limited antimicrobial spectrum and low activity in neutral environments, with the innovation of modification technologies, fermentation processes, and the development of composite formulations, Nisin’s application in the beverage industry will continue to expand. In the future, it will gradually replace some chemical preservatives, becoming a core component of beverage preservation, providing safer, more efficient, and natural solutions for the industry, and promoting the high-quality development of the beverage industry.