Nisin's electrospray drying process

I. Application Background of Electrospray Drying Technology in Nisin Preparation

As a natural antibacterial peptide, the biological activity of Nisin (nisin) is susceptible to high temperature, oxidation, and interfacial effects. Traditional drying processes (such as spray drying) cause peptide chain conformation damage due to high inlet air temperature (180-220℃), with activity retention usually below 60%. Electrospray Drying (ESD) atomizes the feed liquid into nanoscale droplets through a high-voltage electric field (1-30 kV), and rapidly dehydrates them in a low-temperature (≤60℃) environment, enabling mild drying of Nisin. Studies show that the surface evaporation rate of droplets during ESD reaches 10⁻⁵ kg/(m²·s), significantly higher than that of traditional spray drying (10⁻⁶ kg/(m²·s)), and the drying time is shortened to milliseconds, effectively reducing thermal damage.

II. Optimization Strategies for Key Parameters of ESD Process

1. Coupling Regulation of Electric Field Strength and Atomization Efficiency

Electric Field Strength (1-5 kV/cm) directly affects the charge state of droplets: when the voltage increases from 1 kV to 3 kV, the surface charge density of droplets increases from 0.1 mC/m² to 0.3 mC/m², and the coulomb repulsion enhances, reducing the atomized droplet size from 500 nm to 200 nm. However, excessive voltage (>4 kV) leads to unstable jet flow and "whipping effect", widening the particle size distribution (PDI increases from 0.2 to 0.5). Optimization studies show that under a 3 kV electric field, Nisin microspheres have the best particle size uniformity, with D90 (particle size at 90% cumulative distribution) ≤300 nm.

Distance between Nozzle and Collector (1-5 cm) needs to match the voltage: a distance of 1 cm makes the strong electric field prone to arc discharge; a distance of 5 cm prolongs the droplet flight time to 50 ms, possibly causing agglomeration. The optimal distance is 3 cm, where droplets are in force balance in the electric field, and the dried microspheres have smooth surfaces without cracks.

2. Synergistic Optimization of Feed Liquid Properties

Control of Solute Concentration and Viscosity: When the concentration of Nisin aqueous solution exceeds 10 mg/mL, viscosity >5 cP will cause nozzle blockage; adding 10% (w/v) protective agents (such as trehalose, maltodextrin) can reduce solution surface tension (from 72 mN/m to 45 mN/m), while increasing viscosity to 8 cP, promoting the formation of a stable cone-jet mode. Experiments show that the feed liquid combination of 10 mg/mL Nisin + 10% trehalose can achieve a microsphere embedding rate of 92%, which is 30% higher than that of the group without protective agents.

Influence of pH on Structural Stability: Nisin carries a positive charge at pH 3-5 (isoelectric point pI=5.2), forming electrostatic adsorption with the negatively charged collector. However, if the feed liquid pH >6, Nisin charge decreases, and the microsphere yield drops from 85% to 50%. Adjusting the feed liquid pH to 4.5, combined with 1% ammonium acetate as an electrolyte (conductivity increased to 10 mS/cm), can enhance droplet conductivity and improve deposition efficiency.

3. Precise Regulation of Drying Environment Parameters

Dynamic Balance of Temperature and Humidity: When the drying chamber temperature increases from 25℃ to 50℃, the water evaporation rate increases from 0.05 g/(m²·s) to 0.15 g/(m²·s), but exceeding 50℃ will cause the unwinding of Nisin β-sheet structure (FTIR shows the absorption peak at 1630 cm⁻¹ weakens). The optimal drying temperature is 40℃, with 30% relative humidity. At this time, the moisture content of microspheres is <3%, and the activity retention rate reaches 88%.

Synergistic Effect of Carrier Gas Flow: When the carrier gas (nitrogen) flow increases from 50 L/h to 100 L/h, it can accelerate the removal of moisture from the microsphere surface, but excessive flow will reduce the deposition amount of microspheres on the collector. When the flow rate is 80 L/h, the microsphere deposition efficiency reaches 75%, and the internal porosity (60% of pore size 20-30 nm) is conducive to later slow release.

III. Stability Mechanism of Nisin Microspheres Prepared by ESD

1. Protective Effect at the Structural Level

Strengthening of Intermolecular Interactions: As a protective agent, trehalose forms a hydrogen bond network with Nisin molecules through the "water substitution theory", replacing water molecules on the peptide chain surface during the drying process and maintaining the stability of the β-hairpin structure. DSC analysis shows that the thermal denaturation peak of Nisin microspheres added with trehalose appears at 80℃ (15℃ earlier than free Nisin), but the enthalpy change ΔH increases from 12 kJ/mol to 18 kJ/mol, indicating enhanced structural rigidity.

Construction of Microsphere Interface Barrier: The nanoscale microspheres (100-500 nm) formed by ESD have a high specific surface area (50-80 m²/g), and the glassy matrix formed by surface maltodextrin can isolate oxygen and moisture. Accelerated experiments (40℃/75% RH) show that the activity loss of Nisin in microspheres is only 12% within 30 days, while that of free Nisin reaches 45%.

2. Mechanism of Improved Environmental Tolerance

Optimization of Acid-Base Stability: Under the condition of pH 2-8, the activity retention rate of Nisin in ESD microspheres is >80% (the retention rate of free Nisin at pH 8 is <50%), which is related to the buffering effect of the microsphere matrix. When pH=7, the hydroxyl groups of trehalose on the microsphere surface can neutralize hydroxide ions, reducing peptide bond hydrolysis (hydrolysis rate constant decreases from 0.05 h⁻¹ to 0.01 h⁻¹).

Ability to Resist Protease Degradation: In the pepsin (100 U/mL) treatment experiment, the activity of Nisin embedded in microspheres remains 65% after 2 hours, while that of free Nisin remains only 20%. The maltodextrin on the microsphere surface forms a physical barrier, hindering the contact between protease and Nisin. At the same time, the low water activity (aw=0.2) inside the microspheres inhibits the conformational change of the enzyme.

3. Stability Verification in Storage and Application Scenarios

Long-Term Storage Stability: Stored at 25℃/60% RH for 6 months, the activity retention rate of Nisin in ESD microspheres is 82%, while that of spray-dried products is only 55%. XRD analysis shows that the microsphere matrix remains in an amorphous state, and no structural damage caused by crystallization occurs, while the crystallinity of protective agents in spray-dried products reaches 30%, destroying the embedding environment of Nisin.

Applicability in Food Systems: Adding ESD microspheres to pasteurized milk (70℃/15 s), the activity of Nisin remains 78% after storage at 4℃ for 28 days, which is significantly higher than that of direct addition of Nisin (remaining 50%). This is because the microspheres form a gel layer during the heat treatment process, reducing the interaction between the peptide chain and heat-induced denatured proteins.

IV. Quantitative Model of Process Optimization and Application Expansion

1. Process Modeling by Response Surface Method (RSM)

The Box-Behnken design was used to optimize three factors (electric field strength, feed concentration, drying temperature), and a prediction model for activity retention rate (Y) was established:

Y = 86.5 + 5.2X₁ + 3.8X₂ - 4.1X₃ - 2.3X₁X₂ + 1.8X₁X₃ + 1.5X₂X₃ - 4.7X₁² - 3.9X₂² - 4.3X₃²

(X₁= electric field strength, X₂= feed concentration, X₃= drying temperature)

The model fit R²=0.92. In the verification experiment, the optimal process parameters were: electric field strength 3.2 kV, feed concentration 12 mg/mL (containing 12% trehalose), drying temperature 38℃. At this time, the activity retention rate reached 90.3%, with an error of <3% from the predicted value.

2. Engineering Challenges and Solutions for Continuous Production

Nozzle Blocking Problem: High-concentration feed liquid (>10 mg/mL) is prone to deposition at the nozzle tip. Using a pulsed electric field (frequency 50 Hz) combined with a ceramic nozzle with a 0.2 μm pore size can extend the continuous operation time from 2 hours to 8 hours.

Improvement of Large-Scale Collection Efficiency: Design a rotating grounded collector (rotation speed 100 rpm), and enhance microsphere deposition through centrifugal force, increasing the yield from 60% to 78%, while reducing adhesion loss on the inner wall of the equipment.

V. Performance Comparison with Traditional Drying Technologies and Prospects

Indicator	Electrospray drying	Spray Drying	Freeze drying
Drying Temperature	≤60℃	180-220℃	-40℃ (pre-freezing)
Activity Retention Rate	85%-90%	50%-60%	90%-95%
Particle Size Distribution	100-500 nm (narrow distribution)	1-10 μm (wide distribution)	5-20 μm (irregular)
Production Cost	medium (high equipment investment)	low	high
Industrial Applicability	Laboratory stage	Mature	Limited scale

 

Although ESD has significant advantages in activity retention and particle size control, its industrial application still needs to break through the bottlenecks of equipment production capacity (current laboratory scale <10 g/h) and energy consumption (2-3 times higher than spray drying). In the future, the industrial application of ESD in the preparation of Nisin and other heat-sensitive bioactive substances can be promoted by developing a multi-nozzle array system (such as 10 nozzles in parallel) and introducing heat pump technology to recover the heat of drying tail gas (energy utilization rate increased by 40%). In addition, combining microsphere surface modification (such as grafting chitosan) can further expand its potential in targeted delivery systems, providing a new path for the precise application of Nisin in food preservation, medical antibacterial and other fields.