Industrial production optimization of Nisin

The industrial production of Nisin can be optimized through three key directions: precise control of fermentation processes, upgrading of extraction and purification technologies, and improvement of quality control systems. These efforts can achieve a purity of over 95% and control batch-to-batch activity variation within ±5%. The specific optimization pathways are as follows:

I. Fermentation Process Optimization: Enhancing Nisin Yield and Initial Purity at the Source

Nisin is produced by fermentation of Streptococcus lactis. The "strain activity, substrate utilization, and environmental parameters" during fermentation directly determine product purity (the initial purity of Nisin in fermentation broth is typically only 20%–30%, containing large amounts of impurities such as bacterial proteins, lactose, and organic acids). Key optimization steps include:

1. Breeding and Activation of High-Yield Strains

Strain Improvement: High-yield strains are screened using ultraviolet mutagenesis and genetic engineering (e.g., overexpression of the Nisin synthase gene nisA). This increases the Nisin fermentation titer from the traditional 2000–3000 IU/mL to 5000–6000 IU/mL while reducing the secretion of by-products (e.g., bacterial proteins) by 15%–20%, alleviating subsequent purification pressure.

Seed Activation: A "two-stage seed culture" is adopted: primary shake flask culture (MRS medium, 30°C for 18 hours); secondary seed tank culture (optimized medium, 30°C, DO 30%–40%, pH 6.5). This ensures the seed solution reaches a bacterial concentration of over 10⁸ CFU/mL with consistent activity, avoiding batch fluctuations due to uneven seed viability.

2. Optimization of Fermentation Medium: Reducing Impurity Formation

Carbon-Nitrogen Ratio: A composite carbon source of "glucose + lactose" (3:1 ratio) is used—glucose provides rapid energy for initial growth, while lactose is slowly metabolized to sustain Nisin synthesis. For nitrogen sources, "yeast extract + peptone" (2:1 ratio) is selected to avoid raw materials like soy protein that easily produce miscellaneous proteins, reducing miscellaneous protein content in the fermentation broth by 25%.

Precursor and Regulator Addition: 0.1% L-valine (a precursor amino acid for Nisin structure) is added in the early fermentation stage to enhance product synthesis efficiency. In the mid-stage, 0.05% trisodium citrate is added to stabilize the fermentation broth pH (reducing inhibition of bacteria by organic acids) and lower calcium and magnesium ion concentrations (preventing precipitation with Nisin, which would hinder subsequent extraction).

3. Precise Control of Fermentation Process Parameters

Dynamic pH Regulation: An automatic alkali supplementation system (2mol/L NaOH) maintains pH at 6.0–6.5 (the optimal pH for Nisin synthesis). This avoids reduced bacterial enzyme activity at pH <5.5 and contamination risks at pH >7.0, narrowing batch-to-batch Nisin yield variation from ±15% to ±5%.

Dissolved Oxygen (DO) and Temperature Control: In the early fermentation stage (0–8h), DO is controlled at 40%–50% to promote bacterial proliferation; in the late stage (8–24h), DO is reduced to 20%–30% to induce Nisin synthesis. The temperature is maintained at 30±1°C throughout, as fluctuations of ±2°C can reduce Nisin yield by 10%.

Feeding Strategy: A "constant-rate feeding" approach is used (after 12h of fermentation, 20% glucose solution is fed at a rate of 2mL/(L·h)) to prevent bacterial autolysis due to carbon source depletion (autolysis releases large amounts of bacterial proteins, increasing impurities).

II. Upgrading Extraction and Purification Technologies: Enhancing Product Purity

After pretreatment (removal of bacteria and impurities), the fermentation broth undergoes "separation, purification, and refinement" to improve purity. Traditional processes (salting out + ethanol precipitation) achieve only 60%–70% purity, but the following technological upgrades can raise purity to over 95%:

1. Pretreatment: Efficient Removal of Bacteria and Macromolecular Impurities

Microfiltration Clarification: 0.22μm ceramic membrane microfiltration (operating pressure 0.2–0.3MPa, temperature 30°C) replaces traditional plate-frame filtration, achieving 99.9% bacterial removal while retaining Nisin (molecular weight 3.5kDa, which can pass through the membrane), reducing bacterial protein carryover to subsequent processes.

Decolorization and Desalination: A 732-type cation exchange resin (loaded at pH 2.5–3.0, eluted with 2mol/L NaCl) adsorbs Nisin while removing pigments (e.g., carotenoids) and partial inorganic salts (80% desalination rate), increasing fermentation broth transmittance from 60% to 90%.

2. Core Purification: Targeted Enrichment of Nisin

Macroporous Resin Adsorption: XAD-16 macroporous resin (non-polar, with Nisin adsorption capacity of 80mg/g) is used. The fermentation broth is loaded at a flow rate of 2BV/h and eluted with 50% ethanol (containing 0.1mol/L HCl), achieving over 90% Nisin recovery and increasing purity from 30% to 75%.

Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) Refinement: For high-purity products (e.g., food-grade pure Nisin), a C18 column (mobile phase: methanol-0.1% trifluoroacetic acid = 60:40, flow rate 1mL/min) is used to collect the main Nisin peak fraction, achieving 95%–98% purity and removing trace impurity peptides (e.g., the Nisin analog nisin Z).

3. Drying Process: Avoiding Activity Loss

Spray Drying Optimization: Inlet temperature 180–190°C, outlet temperature 70–75°C, atomization pressure 0.3MPa. This avoids Nisin denaturation due to high temperatures (Nisin is easily inactivated at >80°C), achieving over 95% activity retention.

Vacuum Freeze-Drying: For high-value-added products (e.g., pharmaceutical-grade Nisin), vacuum freeze-drying (-50°C pre-freezing, vacuum degree 10Pa, sublimation temperature 30°C) is used, achieving >98% activity retention and better product solubility (no agglomeration due to thermal denaturation).

III. Improvement of Quality Control Systems: Ensuring Batch Stability

Poor batch stability (large fluctuations in activity and purity) is a core challenge in industrial production. A "full-process testing + data traceability" system must be established to control batch-to-batch variation within ±5%:

1. Key Node Testing

Real-Time Fermentation Monitoring: Samples are taken every 2 hours to test Nisin activity (agar diffusion method using Micrococcus luteus as the indicator strain), bacterial concentration (OD600), and pH. Feeding rate and pH are adjusted promptly to avoid abnormal batches.

Purity Testing During Purification: HPLC is used to test Nisin purity (area normalization method) at each step. If purity at any step is below the set threshold (e.g., <70% after resin elution), elution conditions are re-optimized to prevent 不合格 intermediate products from entering subsequent processes.

Finished Product Quality Testing: Each batch is tested for purity (HPLC), activity (IU/mg), moisture content (<5%), and heavy metals (Pb <0.1mg/kg) to ensure compliance with GB 25532-2010 Food Additive: Nisin.

2. Standardized Production and Data Traceability

Standardization of Process Parameters: An SOP for Nisin Industrial Production is developed, specifying allowable fluctuation ranges for key parameters in fermentation, extraction, and drying (e.g., ±1°C for temperature, ±0.1 for pH) to avoid human operation differences.

Data Traceability System: An MES system records "raw material information (medium batch number, strain ID), process parameters (fermentation temperature curve, elution flow rate), and testing data (purity and activity at each step)" for each batch. In case of batch variation, the cause can be quickly identified (e.g., low activity in a batch traced to DO dropping below 15% in late fermentation).

3. Optimization of Storage and Packaging

Packaging Materials: Aluminum-plastic composite bags (with polyethylene lining) are used to block oxygen and moisture (Nisin is prone to oxidative inactivation in humid, oxygen-rich environments). Nitrogen is flushed into packages before sealing (oxygen content <2%).

Storage Conditions: Finished products are stored in a cool, dry place at <25°C with relative humidity <60%. Activity is sampled and tested every 3 months during the shelf life to ensure activity decay rate <5%.

IV. Verification of Industrial Application Effects

Through the above optimizations, the Nisin production indicators of a factory have improved as follows:

Purity: Increased from 60%–70% with traditional processes to over 95% (HPLC detection).

Batch Stability: Batch-to-batch activity variation narrowed from ±15% to ±3%–±5%.

Yield: Fermentation titer increased from 3000 IU/mL to 6000 IU/mL, reducing production costs by 30%.

Impurity Content: Bacterial protein and inorganic salt contents decreased by 80% and 90%, respectively, meeting the EU EC 1333/2008 food additive standards.

The core of "purity improvement" in Nisin industrial production lies in "controlling impurities at the fermentation source + upgrading purification technologies," while "batch stability" depends on "standardization of process parameters + full-process quality control." Through high-yield strain breeding, resin adsorption-HPLC refinement, and MES data traceability, industrial production of high-purity, high-stability Nisin can be achieved, meeting the needs of high-end fields such as food preservation and pharmaceutical antibiosis.