Nisin's membrane separation and purification technology

As a natural antibacterial peptide, Nisin has become a research hotspot due to its efficient and mild characteristics in membrane separation and purification technology. The following analysis is carried out from the technical principle, key process and efficiency improvement strategy:

I. Application Principle of Membrane Separation Technology in Nisin Purification

Core Mechanism of Membrane Separation

Nisin has a molecular weight of about 3.5 kDa and belongs to a small molecule peptide. Membrane separation technologies (such as ultrafiltration, nanofiltration, reverse osmosis) achieve separation through membrane pore size screening effect and charge repulsion:

Ultrafiltration (UF): Membranes with a interception molecular weight of 10-50 kDa are commonly used to remove macromolecular impurities such as bacteria and proteins in the fermentation broth and retain Nisin (because the molecular weight is smaller than the membrane pore size).

Nanofiltration (NF): The membrane pore size is 0.5-2 nm, which can interception inorganic salts and some small molecular impurities, and concentrate Nisin at the same time, realizing desalination and purification simultaneously.

Reverse Osmosis (RO): The membrane pore size is smaller and is used for high-concentration concentration, but the stability of Nisin under high pressure should be noted.

Selection of Membrane Materials and Structures

Material Characteristics: Hydrophilic membranes (such as polyethersulfone PES and polyvinylidene fluoride PVDF) are preferred to reduce membrane pollution caused by the hydrophobic effect of Nisin; negatively charged membranes can reduce the adsorption of positively charged impurities (such as bacterial proteins) through electrostatic repulsion.

Membrane Module Form: Hollow fiber membrane modules are more suitable for industrial-scale separation than flat membranes due to their large specific surface area and strong anti-pollution ability.

II. Typical Process Flow of Membrane Separation and Purification of Nisin

Pretreatment and Ultrafiltration Impurity Removal

After the fermentation broth is removed of bacteria by centrifugation or microfiltration (MF), macromolecular proteins are removed by an ultrafiltration membrane (such as a 10 kDa interception molecular weight), and the purity of Nisin in the filtrate can reach 50%-70%.

Key Controls: Operating pressure 0.1-0.3 MPa, temperature 25-40°C (to avoid Nisin denaturation), and cross-flow rate 1-3 m/s to reduce concentration polarization.

Nanofiltration Concentration and Desalination

The ultrafiltration permeate is concentrated by a nanofiltration membrane (such as a interception molecular weight of 1 kDa), and inorganic salts (such as NaCl in the fermentation broth) are removed at the same time. The Nisin concentration can be increased from 0.5-1 g/L to 5-10 g/L, and the purity reaches 80%-90%.

Process Advantages: The nanofiltration process operates at room temperature to avoid damage to Nisin activity by heat, and the energy consumption is lower than that of traditional evaporation concentration.

III. Key Strategies for Improving Membrane Separation Efficiency

1. Membrane Pollution Control Technology

Membrane Surface Modification:

Graft hydrophilic polymers (such as polyethylene glycol PEG) or anti-fouling coatings to reduce the interaction between Nisin and impurities and the membrane;

Introduce electrostatic repulsion groups (such as sulfonic acid groups) to reduce the adsorption of positively charged proteins (Nisin is positively charged under neutral conditions, and a negatively charged membrane surface needs to be matched to reduce its own adsorption loss).

Optimization of Operating Conditions:

Adopt pulsed cross-flow operation (periodically changing the flow rate) to destroy the filter cake layer on the membrane surface;

Regular online cleaning (CIP): Use mild acid/alkali solutions (such as 0.1 M NaOH) or surfactants (such as Tween-80) to remove pollutants deposited on the membrane surface and restore the flux.

2. Integrated Process to Strengthen Purification Efficiency

Combination of Membrane Separation and Affinity Chromatography:

After ultrafiltration to remove impurities, high-selectivity adsorption is achieved through an affinity membrane (coupled with Nisin-specific binding ligands, such as antibodies or receptor fragments), and the purity can reach more than 95% after elution, which is suitable for the preparation of high-value pharmaceutical-grade Nisin.

Dual Membrane Integration System:

For example, the "ultrafiltration-nanofiltration" tandem process first removes macromolecular impurities through ultrafiltration, and then uses nanofiltration to concentrate and desalt at the same time. Compared with the single membrane process, the Nisin recovery rate is increased from 60%-70% to 85%-90%, and the purity is significantly improved.

3. Intelligent Regulation of Process Parameters

Real-time monitoring of membrane flux, transmembrane pressure (TMP) and Nisin concentration, and automatic adjustment of operating parameters (such as flow rate, pressure) through a feedback system to avoid efficiency degradation caused by membrane pollution.

Predict the membrane pollution trend based on machine learning models, optimize the cleaning cycle, reduce downtime, and improve process continuity.

IV. Technical Challenges and Future Directions

Current Bottlenecks

Adsorption loss of Nisin on the membrane surface (about 5%-10%), especially in the nanofiltration concentration stage;

High-concentration Nisin solutions are prone to form a gel layer, leading to a sharp decrease in membrane flux.

Frontier Exploration

Development of new membrane materials: such as nanocomposite membranes (embedded with nanoparticles such as TiO₂ and graphene) to enhance anti-pollution capabilities;

Bionic membrane design: simulate the selective permeability of biological membranes to improve the separation selectivity of Nisin;

Electrically driven membrane process: combined with the action of an electric field (such as electrodialysis), use the charge characteristics of Nisin to strengthen separation and reduce adsorption loss.

Membrane separation technology shows the advantages of high efficiency and energy saving in Nisin purification. Through membrane material modification, pollution control and process integration, the separation efficiency and product purity can be significantly improved. In the future, it is necessary to further break through the problems of membrane adsorption and flux attenuation, and combine intelligent control with new membrane technologies to promote the industrial application upgrade of Nisin membrane separation and purification.