The combined application of Nisin with other natural preservatives

As a natural antimicrobial peptide produced by the fermentation of Streptococcus lactis, nisin has been widely used for preserving dairy products, meat products, canned foods, and other food items. Its widespread adoption stems from its high efficacy against Gram-positive bacteria (e.g., Staphylococcus and Listeria), excellent safety profile, and degradability by human digestive enzymes. However, nisin has inherent limitations: a narrow antimicrobial spectrum (weak inhibitory activity against Gram-negative bacteria and fungi), reduced activity when affected by food matrices (e.g., fats and proteins), and decreased stability in neutral/alkaline environments.

The combined application of nisin with other natural preservatives (such as plant extracts, organic acids, and enzyme preparations) has emerged as a research hotspot in food preservation. Through a "synergistic enhancement" mechanism, this combination expands the antimicrobial spectrum, improves stability, reduces the dosage of individual preservatives, and avoids the safety risks associated with chemical preservatives. This article explores the synergistic mechanisms underlying such combinations, analyzes the application scenarios and research progress of nisin paired with different types of natural preservatives, and provides references for practical applications in the food industry.

I. Core Synergistic Mechanisms of Nisin Combined with Natural Preservatives

The synergy between nisin and other natural preservatives essentially overcomes the functional limitations of single preservatives and achieves a "1+1>2" antimicrobial effect through three key pathways: "target complementarity," "disruption of microbial defense systems," and "improved environmental adaptability." The specific mechanisms are categorized as follows:

(I) Target Complementarity to Expand Antimicrobial Spectrum

Different natural preservatives act on distinct antimicrobial targets. Nisin exerts its effect primarily by disrupting the cell membrane of Gram-positive bacteria—inserting into the membrane to form pores, which leads to the leakage of intracellular substances. However, it is ineffective against the outer membrane of Gram-negative bacteria, which contains lipopolysaccharides (LPS) that block nisin penetration. In contrast, some natural preservatives (e.g., plant polyphenols and organic acids) target microbial cell wall synthesis, enzyme activity, or genetic material, forming a complementary effect with nisin:

For example, when nisin is combined with ε-polylysine (a cationic peptide produced by Streptomyces), nisin disrupts the cell membrane of Gram-positive bacteria, while ε-polylysine adsorbs to the outer membrane of Gram-negative bacteria and destroys the LPS structure. This allows nisin to penetrate the outer membrane and act on the inner cell membrane. Together, they can simultaneously inhibit Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative), expanding the antimicrobial spectrum from solely Gram-positive bacteria to both groups of pathogens.

Another example: when nisin is combined with carvacrol (a phenolic compound extracted from oregano and thyme), nisin acts on the cell membrane, while carvacrol inhibits the activity of microbial respiratory chain enzymes and blocks energy metabolism. Under this dual action, the inhibition rate against fungi (e.g., yeasts and molds) increases from less than 10% (with nisin alone) to over 60%, addressing nisin’s weak antifungal activity.

(II) Disrupting Microbial Defense Systems to Enhance Antimicrobial Activity

When exposed to a single preservative, microorganisms develop adaptive defenses—such as synthesizing drug-resistant proteins or enhancing cell membrane repair capabilities. The combination of nisin with other natural preservatives disrupts these defenses, thereby improving antimicrobial efficiency:

Some natural preservatives (e.g., organic acids and lysozyme) weaken microorganisms’ ability to repair cell membranes. For instance, when nisin is combined with lactic acid (a natural organic acid), lactic acid lowers the pH of the food matrix. This not only inhibits the growth of acid-sensitive microorganisms but also damages the integrity of Gram-positive bacterial cell membranes, reducing their ability to repair nisin-induced damage (e.g., decreasing the regeneration of membrane phospholipids). As a result, nisin’s minimum inhibitory concentration (MIC) decreases from 256 IU/mL to 64 IU/mL, enhancing its antimicrobial activity by 4-fold.

Plant extracts (e.g., tea polyphenols and allicin) inhibit the expression of microbial drug-resistant genes. For example, Listeria expresses nisin-resistant genes (e.g., the nsr gene, which encodes a nisin-degrading enzyme) when continuously exposed to low doses of nisin. Tea polyphenols, however, inhibit the transcription of the nsr gene, reducing the production of resistant enzymes. This allows nisin to maintain its inhibitory activity against Listeria and prevents the development of resistance.

(III) Improving Environmental Adaptability to Enhance Nisin Stability

Nisin is prone to inactivation in neutral/alkaline environments (pH > 7.0), during high-temperature processing (> 121°C), or in high-fat food matrices. Some natural preservatives enhance nisin stability by "protecting its structure" and "reducing matrix interference":

Polysaccharide-based natural preservatives (e.g., chitosan and pectin) can encapsulate nisin to form microcapsules, minimizing damage from environmental factors. For example, when nisin is complexed with chitosan into microcapsules, its half-life in alkaline meat products (e.g., braised beef, pH 8.0) extends from 2 days to 7 days. This is because the cationic properties of chitosan form a stable complex with nisin, preventing it from being degraded by proteases in the alkaline environment.

Antioxidant natural preservatives (e.g., vitamin E and rosemary extract) reduce nisin oxidation and inactivation. The disulfide bonds in the nisin molecule are prone to breaking under high-temperature or high-oxygen conditions, leading to structural damage. However, rosmarinic acid in rosemary extract scavenges free radicals and protects the stability of disulfide bonds. This allows nisin to retain over 80% of its activity after sterilization at 121°C (compared to only 30% activity retention when nisin is used alone).

II. Research on Nisin Combined with Different Types of Natural Preservatives

Based on the source and characteristics of natural preservatives, the combined application of nisin can be divided into four categories: "plant-derived preservatives," "microbial-derived preservatives," "animal-derived preservatives," and "organic acid preservatives." Different combinations exhibit distinct synergistic effects in various food matrices:

(I) Nisin Combined with Plant-Derived Preservatives

Plant-derived preservatives (e.g., polyphenols, essential oils, and alkaloids) offer broad antimicrobial spectra and abundant sources. When combined with nisin, they significantly expand the antimicrobial range, making them ideal for foods vulnerable to multi-microbial contamination (e.g., fruit and vegetable products, meat products):

Nisin + Tea Polyphenols (tea extract): Tea polyphenols inhibit Gram-negative bacteria (e.g., Salmonella) and fungi (e.g., Penicillium). Their combination with nisin is used for preserving chilled chicken. Studies show that under 4°C refrigeration, a combination of 0.2 g/kg nisin and 0.5 g/kg tea polyphenols reduces the count of Salmonella in chicken by 3 log units within 14 days and inhibits mold growth (nisin alone has no effect on Salmonella). Additionally, the antioxidant properties of tea polyphenols reduce fat oxidation in chicken, extending its shelf life to 21 days (compared to 7 days for the blank group).

Nisin + Carvacrol (oregano essential oil): The lipophilic nature of carvacrol enables it to penetrate microbial cell membranes. Its combination with nisin is used for preserving fruit and vegetable juices. In apple juice, a combination of 0.1 g/kg nisin and 0.05 g/kg carvacrol achieves inhibition rates of 99% against E. coli (Gram-negative) and 95% against yeasts. Under 25°C room temperature storage, the shelf life of apple juice extends from 5 days to 12 days, while avoiding off-flavors caused by chemical preservatives (e.g., sodium benzoate).

Nisin + Berberine (Coptis extract): Berberine inhibits drug-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus [MRSA]). Its combination with nisin is used for preserving baked goods (e.g., bread). Research indicates that a combination of 0.3 g/kg nisin and 0.4 g/kg berberine inhibits MRSA growth in bread, reduces mold contamination, and extends the shelf life from 3 days to 7 days—without affecting the bread’s texture or color.

(II) Nisin Combined with Microbial-Derived Preservatives

Microbial-derived preservatives (e.g., ε-polylysine, natamycin, and subtilin) share similar sources with nisin, offer high safety, and have complementary antimicrobial mechanisms. They are suitable for foods with strict safety requirements (e.g., dairy products, fermented foods):

Nisin + ε-Polylysine: ε-Polylysine inhibits both Gram-negative bacteria and fungi. Its combination with nisin is used for preserving yogurt. In plain yogurt, a combination of 0.15 g/kg nisin and 0.2 g/kg ε-polylysine inhibits E. coli and yeasts that may contaminate yogurt (nisin alone has no effect on either). Under 2–6°C refrigeration, the shelf life of yogurt extends from 21 days to 35 days, without affecting the number of active lactic acid bacteria.

Nisin + Natamycin: Natamycin is a specific antifungal agent. Its combination with nisin addresses nisin’s weak antifungal activity and is used for preserving cheese. In cheddar cheese, a combination of 0.2 g/kg nisin and 0.05 g/kg natamycin simultaneously inhibits Listeria (Gram-positive, a common cause of food poisoning) and Penicillium (fungi, which cause cheese mold). Under 4°C storage, the shelf life of cheese extends from 45 days to 60 days, and the dosage of natamycin is only 1/3 of that required when used alone, reducing costs.

Nisin + Subtilin: Subtilin has a complementary antimicrobial spectrum to nisin against Gram-positive bacteria (e.g., effective against Bacillus cereus). Its combination with nisin is used for preserving canned foods (e.g., canned mushrooms). After sterilization of canned mushrooms at 121°C, a combination of 0.2 g/kg nisin and 0.1 g/kg subtilin completely inhibits the germination of Bacillus cereus (nisin alone has poor spore-inhibiting effects), preventing can swelling and spoilage and extending the shelf life to 12 months.

(III) Nisin Combined with Animal-Derived Preservatives

Animal-derived preservatives (e.g., lysozyme, lactoferrin, and chitin) are naturally safe and easily absorbed by the human body. Their combination with nisin is suitable for foods for special populations (e.g., infant food, health food):

Nisin + Lysozyme (egg white extract): Lysozyme destroys the peptidoglycan structure of bacterial cell walls. Its combination with nisin enhances inhibitory effects against Gram-positive bacteria and is used for preserving infant formula. In formula milk powder, a combination of 0.1 g/kg nisin and 0.3 g/kg lysozyme inhibits Staphylococcus aureus and Listeria monocytogenes that may contaminate the powder. Under 25°C storage, the shelf life of the powder extends from 6 months to 9 months, and both components are natural, meeting the safety requirements for infant food.

Nisin + Lactoferrin (milk extract): Lactoferrin inhibits microbial growth by binding to iron ions (which microorganisms require for metabolism). Its combination with nisin is used for preserving liquid milk. In pasteurized milk, a combination of 0.1 g/kg nisin and 0.2 g/kg lactoferrin inhibits the growth of thermophilic bacteria (e.g., streptococci) in milk. Under 4°C refrigeration, the shelf life of pasteurized milk extends from 7 days to 14 days, and the addition of lactoferrin enhances the nutritional value of milk.

Nisin + Chitin (shrimp and crab shell extract): The film-forming property of chitin encapsulates nisin, improving its stability. Its combination with nisin is used for preserving meat products (e.g., sausages). In pork sausages, a combination of 0.2 g/kg nisin and 0.5 g/kg chitin forms a protective film on the sausage surface, reducing contact between nisin and fat (to avoid nisin inactivation) and inhibiting Salmonella and E. coli in the sausages. Under 4°C storage, the shelf life of the sausages extends from 10 days to 20 days.

(IV) Nisin Combined with Organic Acid Preservatives

Organic acid preservatives (e.g., lactic acid, citric acid, and malic acid) are commonly used in the food industry. They inhibit microbial growth by lowering pH, and their combination with nisin enhances inhibitory effects against acid-tolerant bacteria. They are suitable for acidic foods (e.g., pickles, beverages):

Nisin + Lactic Acid: Lactic acid lowers the pH of food and enhances nisin’s activity against Gram-positive bacteria. Its combination with nisin is used for preserving pickles. In Chinese cabbage pickles, a combination of 0.15 g/kg nisin and 1.0 g/kg lactic acid inhibits Listeria (highly acid-tolerant, poorly inhibited by lactic acid alone). The sour taste of lactic acid also improves the pickle’s flavor. Under 25°C storage, the shelf life of the pickles extends from 15 days to 30 days, while avoiding the use of nitrites.

Nisin + Citric Acid: Citric acid chelates metal ions (e.g., Ca²⁺ and Mg²⁺, which maintain cell membrane stability) in microbial cells. Its combination with nisin destroys cell membrane integrity and is used for preserving juice beverages (e.g., orange juice). In fresh-squeezed orange juice, a combination of 0.1 g/kg nisin and 0.8 g/kg citric acid inhibits E. coli and yeasts. Under 4°C refrigeration, the shelf life of orange juice extends from 3 days to 8 days, and the addition of citric acid enhances the juice’s flavor.

Nisin + Malic Acid: Malic acid has a mild acidity. Its combination with nisin is used for preserving snack foods (e.g., jelly, candy). In strawberry jelly, a combination of 0.1 g/kg nisin and 0.6 g/kg malic acid inhibits Staphylococcus aureus and molds. Under 25°C storage, the shelf life of the jelly extends from 10 days to 20 days, and the fruity flavor of malic acid improves the jelly’s taste.

III. Challenges and Optimization Directions for Combined Application

Despite the significant advantages of combining nisin with natural preservatives, practical industrial applications face challenges such as "matrix-dependent synergistic effects," "high costs," and "lack of standardization." Optimization is needed in the following areas:

(I) Optimizing Combination Schemes for Specific Food Matrices

Different food matrices (e.g., fat content, pH, water activity) significantly affect the synergistic effect of nisin and natural preservatives. For example, in high-fat meat products (e.g., fatty sausages), fat adsorbs nisin and plant essential oils, reducing their antimicrobial activity; in acidic fruit and vegetable juices, low pH enhances the synergy between organic acids and nisin. Future research should focus on "matrix-synergy" correlation studies for specific food categories (e.g., dairy, meat, fruit and vegetable products) to develop personalized combination schemes. For instance, microencapsulation technology (e.g., chitosan-encapsulated nisin and carvacrol) can be used in high-fat foods to reduce preservative adsorption by fat and maintain synergistic activity.

(II) Reducing the Cost of Combined Application

The high production cost of some natural preservatives (e.g., tea polyphenols, lactoferrin) makes nisin combination schemes more expensive than chemical preservatives (e.g., sodium benzoate, potassium sorbate), limiting their application in mid-to-low-end foods. Future efforts should reduce the cost of natural preservatives through "source expansion" (e.g., extracting plant polyphenols from agricultural waste such as tea residues and grape skins) and "fermentation process optimization" (e.g., using genetic engineering to modify strains and increase ε-polylysine yield). Additionally, "precision dosage optimization" (e.g., using response surface methodology to screen for minimum effective dose combinations) can reduce the total preservative dosage while ensuring preservation efficacy, thereby lowering costs.

(III) Establishing a Standardized Evaluation System

Currently, there is no unified standard for evaluating the combined application of nisin and natural preservatives. For example, some studies use "inhibition zone diameter" to evaluate synergy, while others use "microbial count reduction rate," making it difficult to compare results across studies. A standardized evaluation system covering "synergistic mechanisms, antimicrobial efficacy, safety, and sensory impact" should be established. For instance, formulating a Guideline for Evaluating the Combined Application of Nisin and Natural Preservatives to clarify synergy criteria (e.g., a fractional inhibitory concentration [FIC] index < 0.5 for strong synergy) and safety testing indicators (e.g., acute toxicity, genotoxicity) will promote the standardization of combined applications.

The combined application of nisin with other natural preservatives overcomes nisin’s limitations of a narrow antimicrobial spectrum and poor stability through synergistic mechanisms of target complementarity, microbial defense system disruption, and improved environmental adaptability. It shows broad application prospects in dairy products, meat products, fruit and vegetable products, and other fields. Different combinations (e.g., nisin + tea polyphenols, nisin + ε-polylysine, nisin + lysozyme) can be flexibly selected based on food matrix characteristics and preservation requirements, achieving the dual goals of "natural safety" and "high-efficiency preservation."

Future efforts should address the practical challenges of combined applications by optimizing schemes for specific food matrices, reducing costs, and establishing standardized systems. This will promote the large-scale application in the food industry and provide consumers with safer and longer-shelf food products.