Ultrasonic-assisted extraction of Nisin

I. Foundation of Process Optimization: Ultrasonic Extraction Principles and Nisin Characteristics

Nisin, a polypeptide antibiotic secreted by Streptococcus lactis, has a molecular weight of approximately 3.5 kDa, featuring thermal stability (retaining 80% activity after autoclaving at 121°C for 15 minutes) and pH sensitivity (high solubility under acidic conditions). Ultrasonic-assisted extraction disrupts microbial cell walls through cavitation effects (local high temperature and pressure from instantaneous bubble collapse in liquids) and mechanical vibration (mechanical shearing force at 20–100 kHz), promoting Nisin release. Its core advantages include:

Shortened extraction time (traditional soaking requires 2–4 hours, while ultrasonic extraction reduces it to 30–60 minutes);

Improved mass transfer efficiency (micro-jet flow generated by cavitation bubble collapse enhances solute diffusion);

Low-temperature operation to protect activity (temperature rise ≤10°C during ultrasonication avoids polypeptide denaturation).

II. Optimization Strategies for Key Process Parameters

(1) Regulation of Ultrasonic Physical Parameters

Power Density: The optimal range is typically 200–400 W/L. Power below 150 W/L yields weak cavitation and low cell wall 破碎效率 (breakage efficiency), while exceeding 500 W/L causes abrupt temperature rise, leading to Nisin molecular disulfide bond cleavage (activity loss >15%). Example: Extracting from skim milk fermentation broth at 300 W/L increases Nisin yield by 42% compared to traditional methods.

Ultrasonic Time: Follow the "segmented pulse" principle to avoid energy decay from continuous ultrasonication. Recommended parameters: 30s ultrasound + 15s interval, total time ≤45 minutes. Exceeding 60 minutes reduces yield by 10%–15% due to weakened shearing force from excessive cavitation bubble expansion.

Frequency Selection: Low frequency (20–30 kHz) suits cell wall breaking, while high frequency (80–100 kHz) facilitates solute diffusion. Composite frequency (e.g., alternating 25 kHz and 80 kHz) balances efficiency and activity, increasing Nisin purity to ≥92%.

(2) Optimization of Extraction System Environment

pH Regulation: Nisin has an isoelectric point at pH 5.0 and dissolves easily with positive charges under acidic conditions. The optimal extraction pH is 3.5–4.5, achieving a solubility of 1.2 g/L (only 0.3 g/L at pH 7.0). Adding 0.1–0.2 M citric acid-sodium citrate buffer maintains acidity and inhibits contaminant growth.

Temperature Co-control: Each 1°C temperature rise during ultrasonication causes ~3% Nisin activity loss. Use circulating cooling water to maintain 25–30°C or perform ultrasonication in an ice bath (0–4°C). The latter reduces extraction rate but retains 98% activity.

Solid-Liquid Ratio and Additives: The suitable solid-liquid ratio (based on wet weight of fermented cells) is 1:10–1:15 (g/mL). Adding 0.5% (w/v) non-ionic surfactants like Triton X-100 disrupts the cell membrane lipid bilayer via solubilization, increasing Nisin release by 30%.

III. Experimental Design and Validation Methods for Process Optimization

(1) Response Surface Methodology (RSM) Optimization Model

Using Box-Behnken design with ultrasonic power (A), time (B), and pH (C) as independent variables and Nisin yield (Y) as the response value, a quadratic regression equation is established:

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Y = β₀ + β₁A + β₂B + β₃C + β₄AB + β₅AC + β₆BC + β₇A² + β₈B² + β₉C²

ANOVA analysis identifies significant factors. Typical optimization results: power 320 W/L, time 40 min, pH 3.8, predicted yield 48.7 mg/L, and actual yield 47.2 mg/L in validation experiments, with model fit R² > 0.95.

(2) Activity Retention Evaluation

Biological Potency Assay: Using the cup-plate method (ISO 14469:2001) with Staphylococcus aureus ATCC 25923 as the indicator bacterium, the inhibition zone diameter of the ultrasonic extract at 25°C is compared with standards, showing activity retention ≥90% under optimized processes.

Structure Characterization: Circular dichroism (CD) spectroscopy detects β-sheet structure content. The characteristic absorption peak intensity of Nisin extracted by the optimized process at 218 nm matches the standard, confirming intact secondary structure.

IV. Considerations for Process Scaling and Industrialization

Equipment Selection: Industrial ultrasonic extraction requires tank reactors (volume ≥500 L) with titanium alloy transducers (frequency 20–25 kHz) and temperature control systems, ensuring power density uniformity error ≤±5%.

Energy Consumption and Cost: Ultrasonic extraction increases energy consumption by 15%–20% but reduces comprehensive production costs by >25% due to shortened extraction time and improved yield (calculated for 100 tons/year Nisin production, equipment payback period ~1.5 years).

Downstream Purification Integration: Ultrasonic extract may contain cell wall fragments, requiring pretreatment with centrifugation (8000×g, 15 min) or membrane filtration (0.22 μm microfiltration membrane) to avoid affecting subsequent affinity chromatography or reverse-phase chromatography purification efficiency.

V. Expansion of Cutting-Edge Technology Combination

Ultrasonic-Microwave Synergistic Extraction: Microwave (2450 MHz) assistance enhances molecular thermal motion, synergizing with ultrasonic cavitation to further increase Nisin yield by 12%–18%, but microwave power should be controlled ≤300 W to prevent overheating.

Enzymatic-Ultrasonic Coupling Process: First use lysozyme (500 U/mL) to enzymatically hydrolyze cell walls at 37°C for 30 min, then perform ultrasonic extraction, suitable for high-concentration bacterial systems (wet weight ≥20 g/L) with yields ≥55 mg/L.

Low-Temperature Ultrasonic-Supercritical CO₂ Extraction Integration: Ultrasonic extraction under supercritical conditions (35°C, 10 MPa) utilizes CO₂ solubility and ultrasonic cell wall breaking to simultaneously extract Nisin and remove salts, achieving purity ≥95%.