The future development trends and challenges of Nisin

As a natural antimicrobial peptide produced by the fermentation of Streptococcus lactis, nisin has been widely used in the preservation of dairy products, meat products, canned foods, and other foods. It exhibits high efficiency against Gram-positive bacteria (e.g., Staphylococcus, Listeria), high safety, and degradability by human digestive enzymes. However, nisin has limitations, including a narrow antimicrobial spectrum (weak inhibition against Gram-negative bacteria and fungi), reduced activity affected by food matrices (e.g., fat, protein), and decreased stability in neutral/alkaline environments.

The combined application of nisin with other natural preservatives (e.g., plant extracts, organic acids, enzyme preparations) has become a research hotspot in the field of food preservation. Through a "synergistic enhancement" mechanism, this combination expands the antimicrobial spectrum, improves stability, reduces the dosage of single preservatives, and avoids safety risks associated with chemical preservatives. This article analyzes the application scenarios and research progress of nisin combined with different types of natural preservatives from the perspective of synergistic mechanisms, providing references for food industry practices.

I. Core Synergistic Mechanisms of Combined Nisin and Natural Preservatives

The synergy between nisin and other natural preservatives essentially breaks through the functional limitations of single preservatives and achieves a "1+1>2" antimicrobial effect through three 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 Coverage

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, leading to intracellular substance leakage), but 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, organic acids) act on microbial cell wall synthesis, enzyme activity, or genetic material, forming target complementarity 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—allowing nisin to penetrate the outer membrane and act on the cell membrane. This synergy enables simultaneous inhibition of Staphylococcus aureus (Gram-positive) and E. coli (Gram-negative), expanding the antimicrobial spectrum from a single focus on Gram-positive bacteria to both pathogenic groups.

Another example: when nisin is combined with carvacrol (a phenolic substance 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, 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 (e.g., synthesizing drug-resistant proteins, enhancing cell membrane repair capabilities). The combination of nisin with other natural preservatives disrupts this defense system and improves antimicrobial efficiency:

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

Plant extracts (e.g., tea polyphenols, 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 exposed to low-dose nisin long-term. Tea polyphenols inhibit the transcription of the nsr gene, reducing the production of resistant enzymes and ensuring nisin maintains its inhibitory activity against Listeria, preventing the development of resistance.

(III) Improving Environmental Adaptability to Enhance Nisin Stability

Nisin is prone to inactivation in neutral/alkaline environments (pH > 7.0), high-temperature processing (>121°C), or 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, pectin) can encapsulate nisin to form microcapsules, reducing environmental damage to nisin. 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 nisin from being degraded by proteases in the alkaline environment.

Antioxidant natural preservatives (e.g., vitamin E, 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. Rosmarinic acid in rosemary extract scavenges free radicals and protects disulfide bond stability, allowing nisin to retain over 80% of its activity after sterilization at 121°C (compared to only 30% activity retention with nisin alone).

II. Research on Combined Application of Nisin 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 differentiated synergistic effects in food matrices:

(I) Combined Application of Nisin with Plant-Derived Preservatives

Plant-derived preservatives (e.g., polyphenols, essential oils, alkaloids) have the advantages of a broad antimicrobial spectrum and abundant sources. When combined with nisin, they significantly expand the antimicrobial range, making them suitable 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 chilled chicken preservation. Studies show that under 4°C refrigeration, a combination of 0.2 g/kg nisin + 0.5 g/kg tea polyphenols reduces Salmonella counts 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 chicken fat oxidation, extending the shelf life to 21 days (compared to 7 days for the blank group).

Nisin + Carvacrol (oregano essential oil): The lipophilic nature of carvacrol allows it to penetrate microbial cell membranes. Its combination with nisin is used for fruit and vegetable juice preservation. In apple juice, 0.1 g/kg nisin + 0.05 g/kg carvacrol achieves 99% and 95% inhibition rates against E. coli (Gram-negative) and yeasts, respectively. Under 25°C room temperature storage, the shelf life of apple juice extends from 5 days to 12 days, 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 baked goods (e.g., bread) preservation. Research indicates that 0.3 g/kg nisin + 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 bread texture or color.

(II) Combined Application of Nisin with Microbial-Derived Preservatives

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

Nisin + ε-Polylysine: ε-Polylysine inhibits Gram-negative bacteria and fungi. Its combination with nisin is used for yogurt preservation. In plain yogurt, 0.15 g/kg nisin + 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 cheese preservation. In cheddar cheese, 0.2 g/kg nisin + 0.05 g/kg natamycin simultaneously inhibits Listeria (Gram-positive, a common cause of food poisoning) and Penicillium (fungi, causing 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 used alone, reducing costs.

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

(III) Combined Application of Nisin with Animal-Derived Preservatives

Animal-derived preservatives (e.g., lysozyme, lactoferrin, 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 inhibition against Gram-positive bacteria and is used for infant formula preservation. In formula milk powder, 0.1 g/kg nisin + 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 infant food safety requirements.

Nisin + Lactoferrin (milk extract): Lactoferrin inhibits microbial growth by binding iron ions (required for microbial metabolism). Its combination with nisin is used for liquid milk preservation. In pasteurized milk, 0.1 g/kg nisin + 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 meat product (e.g., sausage) preservation. In pork sausages, 0.2 g/kg nisin + 0.5 g/kg chitin forms a protective film on the sausage surface, reducing contact between nisin and fat (avoiding nisin inactivation) and inhibiting Salmonella and E. coli in sausages. Under 4°C storage, the shelf life of sausages extends from 10 days to 20 days.

(IV) Combined Application of Nisin with Organic Acid Preservatives

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

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

Nisin + Citric Acid: Citric acid chelates metal ions (e.g., Ca²⁺, Mg²⁺, which maintain cell membrane stability) in microbial cells. Its combination with nisin destroys cell membrane integrity and is used for juice beverage (e.g., orange juice) preservation. In fresh-squeezed orange juice, 0.1 g/kg nisin + 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 orange juice flavor.

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

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 natural preservative costs 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., response surface methodology to screen minimum effective dose combinations) can reduce total preservative dosage while ensuring preservation efficacy, 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., FIC index < 0.5 for strong synergy) and safety testing indicators (e.g., acute toxicity, genotoxicity), promoting the standardization of combined applications.

The combined application of nisin with other natural preservatives breaks through 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 needs, achieving the dual goals of "natural safety" and "high-efficiency preservation."

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