The Application of Nisin in Fruit juice beverages

Nisin, a natural antibacterial peptide produced by the fermentation of Lactococcus lactis, is an internationally recognized GRAS (Generally Recognized as Safe)-grade food preservative. It features a narrow antibacterial spectrum, high temperature resistance, easy degradability, and no residue. In the preservation of fruit juice beverages, it mainly inhibits lactic acid bacteria and yeasts—microorganisms that cause product spoilage—by disrupting microbial cell membrane structures and interfering with metabolic pathways. This delays fruit juice spoilage, extends shelf life, while ensuring the product’s naturalness and safety.

I. Spoilage Characteristics and Hazards of Lactic Acid Bacteria and Yeasts in Fruit Juice Beverages

Fruit juice beverages are rich in carbohydrates (fructose, glucose), vitamins, and water, with a pH typically ranging from 3.0 to 4.5 (acidic environment). Although this nutrient-dense, acidic system inhibits most Gram-negative bacteria (e.g., Escherichia coli), it serves as an "ideal breeding ground" for lactic acid bacteria and yeasts—the core microorganisms responsible for fruit juice spoilage:

1. Spoilage Mechanism of Lactic Acid Bacteria

Common spoilage-causing lactic acid bacteria in fruit juices include Lactobacillus plantarum and Lactobacillus brevis. These bacteria tolerate the acidic environment of fruit juices and ferment sugars in the juice to produce organic acids such as lactic acid and acetic acid, further lowering the juice’s pH (e.g., from 4.0 to below 3.0)—a phenomenon known as "excessive acidification." This manifests as the juice’s taste changing from sweet-sour to tart and pungent, with potential off-odors (e.g., fermented or rancid smells). Some lactic acid bacteria also decompose polysaccharides like pectin and cellulose in the juice, causing texture deterioration such as stratification, sedimentation, and reduced viscosity, seriously impairing the product’s sensory quality.

2. Spoilage Mechanism of Yeasts

Typical spoilage-causing yeasts in fruit juices include Saccharomyces cerevisiae and Candida spp. They reproduce rapidly under acidic conditions and ferment sugars via anaerobic respiration to produce ethanol (alcohol) and carbon dioxide, triggering "alcoholic fermentation" in the juice. This results in bubbles (gassiness) and an alcoholic taste; prolonged spoilage further produces undesirable flavor compounds such as acetaldehyde and higher alcohols, stripping the juice of its original fruity aroma. Additionally, yeast metabolites may react with phenolic substances in the juice, causing darkening of color (e.g., apple juice turning from pale yellow to brownish yellow). Massive yeast proliferation also forms flocculent sediment, destroying the juice’s clarity.

These two types of microorganisms are widespread in contamination sources: they may originate from raw materials (e.g., bacteria-laden fruit peels, damaged pulp), processing equipment (e.g., inadequately cleaned presses, filling lines), or the environment (e.g., workshop air, operators’ hands). Once colonized in the juice, they typically cause spoilage within 2–5 days under room temperature storage (25–30℃), drastically shortening the juice’s shelf life.

II. Core Mechanisms of Nisin’s Inhibition on Lactic Acid Bacteria and Yeasts

Nisin’s antibacterial activity primarily targets Gram-positive bacteria, exerting direct and efficient inhibition on lactic acid bacteria (Gram-positive). For yeasts (eukaryotes), inhibition relies on "synergistic effects" or "environmental regulation." The core mechanisms revolve around microbial cell membrane disruption and metabolic interference:

1. Direct Inhibition Mechanism on Lactic Acid Bacteria

As Gram-positive bacteria, lactic acid bacteria have a relatively loose peptidoglycan layer (the main component of the cell wall) outside their cell membranes. Nisin penetrates the cell wall to act on the cell membrane through two pathways:

Membrane perforation effect: Amino acid residues such as lysine and isoleucine in the nisin molecule bind to phospholipids (e.g., phosphatidylglycerol) on the lactic acid bacterial cell membrane and lipid II (a cell wall precursor unique to Gram-positive bacteria) in the cell wall, forming transmembrane channels (with a pore size of approximately 1–2 nm). This increases membrane permeability, causing massive leakage of essential intracellular substances (e.g., potassium ions, amino acids) while allowing harmful substances (e.g., organic acids) from the outside to enter the cell. Ultimately, this leads to osmotic imbalance and metabolic disorder, resulting in the death of lactic acid bacteria.

Interference with cell wall synthesis: The binding of nisin to lipid II competitively inhibits lipid II’s involvement in peptidoglycan synthesis, preventing the normal assembly of the lactic acid bacterial cell wall. The loss of cell wall integrity causes the cell to lyse under external pressure, fundamentally blocking the proliferation and metabolism of lactic acid bacteria.

This dual mechanism gives nisin extremely strong inhibitory effects on lactic acid bacteria in fruit juices. Even at low concentrations (e.g., 50–100 IU/mL), it can significantly reduce the colony count of lactic acid bacteria (e.g., from 10⁵ CFU/mL to below 10² CFU/mL), effectively preventing excessive acidification and texture deterioration of the juice.

2. Synergistic Inhibition Mechanism on Yeasts

The cell membrane structure of yeasts (containing sterols) differs significantly from that of Gram-positive bacteria, making it difficult for nisin to penetrate directly. When used alone, nisin has weak inhibitory effects on yeasts; however, its inhibitory capacity is significantly enhanced through synergy with the acidic environment of the juice or other preservation methods (e.g., low temperature, low oxygen). The key mechanisms are as follows:

Acidic environment enhances permeability: The low pH (3.0–4.5) of fruit juice slightly increases the permeability of the yeast cell membrane. Although nisin cannot form transmembrane channels under this condition, it can adsorb to the membrane surface and interfere with the function of proton pumps (which maintain pH balance inside and outside the cell). This leads to the accumulation of acidic substances inside the yeast cell, inhibiting the activity of respiratory enzymes and slowing down energy metabolism and proliferation.

Synergistic enhancement with low temperature/low oxygen: Under refrigerated storage (0–4℃) or modified atmosphere packaging (filled with nitrogen or carbon dioxide), the metabolic activity of yeasts is already low. Nisin further inhibits enzyme systems on the yeast cell membrane (e.g., hexokinase, involved in sugar metabolism), extending the yeast’s lag phase from 12 hours to over 48 hours. When combined with small amounts of food-grade organic acids (e.g., citric acid, sorbic acid), the organic acids disrupt the yeast cell wall structure, creating conditions for nisin to exert its effect and achieving a "1+1>2" antibacterial result.

III. Application Methods and Preservation Effects of nisin in Fruit Juice Beverages

In the production of fruit juice beverages, the application of nisin must be tailored to the juice type (clear juice, cloudy juice), processing technology (hot filling, cold filling), and shelf life requirements. Rational addition methods and dosages maximize its preservation effects, as detailed below:

1. Application Methods and Dosage Control

Timing of addition: Nisin is typically added during the cooling phase (temperature reduced to 40–50℃) after juice pasteurization and before filling. This avoids inactivation by high temperatures (e.g., 85–90℃ during pasteurization)—though nisin is heat-resistant (retaining 80% activity after 30 minutes of sterilization at 121℃), prolonged exposure to high temperatures reduces its stability. For cold-filled juices, nisin is added directly after filtration and clarification to ensure uniform dispersion.

Dosage: Based on the juice’s contamination risk and shelf life requirements, the dosage is usually controlled at 50–200 IU/mL (International Units per milliliter). Clear juices (e.g., apple juice, orange juice) have lower microbial contamination risks (as filtration removes some microorganisms), requiring 50–100 IU/mL. Cloudy juices (e.g., kiwi juice, strawberry juice) contain pulp particles that easily adhere to microorganisms, requiring 100–200 IU/mL; they are often combined with 0.05%–0.1% potassium sorbate to enhance inhibitory effects on yeasts.

Precautions: As a peptide,nisin is easily degraded by proteases in the juice (e.g., natural fruit proteases). Thus, it should not be mixed directly with unsterilized raw materials and must be added after juice pasteurization (to inactivate proteases). Additionally,nisin loses efficacy in alkaline environments, so the juice’s pH must be stably maintained within the acidic range of 3.0–4.5.

2. Key Manifestations of Preservation Effects

Extended shelf life: Under room temperature storage (25℃), fresh-squeezed juice without nisin typically has a shelf life of only 2–5 days. In contrast, juice with 100 IU/mL nisin shows significantly inhibited proliferation of lactic acid bacteria and yeasts, extending the shelf life to 15–20 days. When combined with refrigeration (4℃), the shelf life can be further extended to 30–45 days—equivalent to the effect of chemical preservatives (e.g., sodium benzoate) but more aligned with consumer demand for "natural preservatives."

Maintained sensory quality: Nisin effectively prevents lactic acid bacteria from producing acids and yeasts from generating alcohol and carbon dioxide, ensuring the juice maintains a stable pH (fluctuation ≤ 0.3) and sweet-sour taste throughout its shelf life, with no off-odors (e.g., fermented or alcoholic smells). It also avoids pectin decomposition-induced stratification and sedimentation, preserving the juice’s clarity (for clear juices) or uniform turbidity (for cloudy juices). The color remains bright (e.g., the orange color of orange juice, the purple-red color of grape juice).

Guaranteed food safety: By inhibiting spoilage-causing lactic acid bacteria and yeasts, nisin reduces harmful metabolites (e.g., excessive organic acids, higher alcohols). It is degraded into amino acids by proteases in the human digestive tract, leaving no residues or toxicity and without altering the juice’s nutrients (e.g., the content of vitamin C and polyphenols remains largely unaffected), aligning with the "natural and healthy" positioning of fruit juice beverages.

IV. Key Precautions and Optimization Directions in Application

Despite nisin’s significant advantages in fruit juice preservation, attention must be paid to the following issues to ensure stable efficacy and compliance:

1. Strict Adherence to Usage Regulations

According to China’s National Food Safety Standard for the Use of Food Additives (GB 2760), the maximum dosage of nisin in fruit and vegetable juice beverages is 0.2 g/kg (approximately 200 IU/mL). Dosage must be strictly controlled to avoid excess—though nisin is highly safe, overuse may cause a slight bitter taste in the juice, affecting flavor. Additionally, nisin only inhibits Gram-positive bacteria and some synergistically inhibited yeasts; it cannot inhibit molds (e.g., Penicillium, Aspergillus). If the juice is at risk of mold contamination, nisin must be combined with other compliant preservatives (e.g., natamycin, specifically for mold inhibition).

2. Adaptation to Juice Matrix Characteristics

Sugars, organic acids, and polyphenols in the juice may affect nisin’s activity:

High-concentration sugars (e.g., concentrated juice before dilution, sugar content > 20%) reduce nisin’s solubility. Nisin should first be dissolved in a small amount of sterile water before addition.

Polyphenols (e.g., chlorogenic acid in apple juice, tannins in grape juice) may bind to nisin, weakening its antibacterial activity. To address this, the nisin dosage can be appropriately increased (within standard limits), or excess polyphenols can be removed during processing via adsorption (e.g., using activated carbon).

3. Enhancing Efficacy through Process Optimization

Nisin’s preservation effect relies on integration with sound processing techniques:

Raw material pretreatment: Remove microorganisms from fruit surfaces via rinsing with clean water and soaking in sodium hypochlorite (50–100 mg/L).

Equipment cleaning: Use CIP (Clean-in-Place) systems to thoroughly clean processing equipment, avoiding microbial contamination from residual substances.

Filling process: Adopt aseptic cold filling technology to reduce secondary contamination. Only by minimizing the initial microbial count (e.g., < 10³ CFU/mL) can nisin’s antibacterial effect be maximized, preventing "inhibition failure" due to excessively high initial contamination.

By directly inhibiting spoilage-causing lactic acid bacteria and synergistically inhibiting yeasts,nisin effectively resolves fruit juice spoilage issues such as acidification, gassiness, and sensory deterioration, extends shelf life, and offers the advantages of naturalness, safety, and no residue. It is an ideal preservative choice for fruit juice beverages amid the "clean label" trend (reducing chemical preservative use). Future research can further explore compound formulations of nisin with natural plant extracts (e.g., rosemary extract, tea polyphenols) to expand the antibacterial spectrum (inhibiting molds simultaneously) and reduce nisin dosage, further enhancing the naturalness and safety of fruit juices.