The fermentation process of Nisin has been improved

I. Core Directions for Fermentation Process Improvement

Nisin, a natural antibacterial peptide produced by Lactococcus lactis fermentation, requires multi-dimensional optimization of strain characteristics, medium composition, and fermentation conditions to break through yield bottlenecks.

II. Key Improvement Strategies and Yield Enhancement Mechanisms

(1) Screening and Modification of High-Yield Strains

Traditional Mutagenesis Breeding: Treating wild-type strains with mutagens (UV, nitrosoguanidine, NTG) to screen mutants with optimized metabolic pathways (e.g., 解除 product feedback inhibition or enhanced precursor synthesis), increasing Nisin yield by 10%-30%.

Genetic Engineering Modification:

Overexpressing Nisin synthesis-related genes (nisA, nisB, nisC) to enhance peptide chain synthesis and modification efficiency;

Knocking out competitive metabolic pathway genes (e.g., lactate dehydrogenase) to reduce carbon flow to by-products like lactate, redirecting energy to Nisin synthesis;

Introducing exogenous transporter genes to promote extracellular Nisin secretion and reduce feedback inhibition from intracellular accumulation.

(2) Medium Composition Optimization

Carbon Source Regulation:

Using a glucose-lactose mixed carbon source (initial glucose 10-15 g/L, followed by lactose fed-batch), leveraging glucose for rapid fermentation initiation and lactose for mid-late synthesis maintenance, extending Nisin production period and increasing yield by 20%-40%.

Exploring alternative carbon sources (starch hydrolysate, inulin) to replace glucose, reducing costs and regulating osmotic pressure for metabolic balance.

Nitrogen Source Optimization:

Complex nitrogen sources (peptone + yeast extract + ammonium sulfate) outperform single sources. Peptone provides essential amino acids (leucine, valine as Nisin precursors), while yeast extract supplies vitamins/nucleotides, optimizing ratios to boost yield by 15%-30%.

Controlling ammonium ion concentration: Excess NH₄⁺ inhibits Nisin synthase activity. Urea fed-batch releases NH₃ slowly to maintain NH₄⁺ at 0.5-1.0 g/L.

Addition of Key Trace Elements and Precursors:

Mn²⁺ (0.1-0.5 mM) activates Nisin synthase, and Cu²⁺ (0.01-0.05 mM) enhances membrane permeability for product secretion;

Exogenous addition of Nisin precursor amino acids (leucine, alanine) bypasses rate-limiting steps, directly participating in peptide assembly to increase yield by 10%-20%.

(3) Fermentation Process Control Strategies

Dynamic pH Regulation:

Optimal pH for Nisin synthesis is 6.0-6.5. Early fermentation uses strain-produced lactic acid to naturally lower pH to 5.5-6.0, while mid-late stages maintain pH at 6.2-6.5 via NaOH/ammonia fed-batch, avoiding acid inhibition and increasing yield by 30%-50%.

Dissolved Oxygen (DO) Optimization:

Maintain DO≥30% in the early stage (0-12 h) for cell proliferation, reduce DO to 10%-20% in the mid-stage (12-24 h) to induce Nisin synthesis (anaerobic/microaerobic environments promote synthase expression), and moderately increase DO to 25% in the late stage (after 24 h) for product secretion, enhancing yield by 25%-40%.

Fermentation Mode Improvement:

Fed-batch fermentation: Start feeding carbon (glucose) and nitrogen (peptone hydrolysate) at the late exponential phase (OD600=5-8) to maintain low substrate concentration, extending the synthesis period and increasing yield by 40%-60% compared to batch fermentation;

Two-phase fermentation system: Adding hydrophobic carriers (n-hexadecane) extracts Nisin into the organic phase, relieving extracellular product inhibition and reducing protease degradation, increasing yield by 30%-50%.

(4) Combined Metabolic Regulation and Fermentation Engineering

Transcriptomics and Metabolomics Guidance: Omics analysis identifies rate-limiting pathways (methionine metabolism, fatty acid synthesis). Targeted gene knockout/overexpression, e.g., enhancing methionine synthesis to provide more S-adenosylmethionine (SAM, Nisin modification cofactor), increases yield by 15%-25%.

High-Cell-Density Fermentation: Using high-cell-density culture (HCD) with optimized osmotic pressure (glycerol) and by-product (lactate) control (calcium carbonate neutralization), cell concentration (OD600) increases from 10-15 to 30-40. Combined with efficient secretion systems, Nisin yield reaches 5-8 g/L, a 50%-100% improvement over traditional processes.

(5) Downstream Process Integration for Yield Enhancement

Pretreating fermentation broth with membrane filtration (ultrafiltration + microfiltration) to remove cells and macromolecular proteins, reducing purification load;

Using affinity chromatography/reverse-phase chromatography combined with pH-induced precipitation (pH 2.0-2.5) to achieve recovery rates >85%, indirectly boosting overall yield.

III. Industrial Challenges and Future Trends

Current bottlenecks include genetic stability of high-yield strains (mutation prone during long-term passage) and metabolic regulation consistency in large-scale fermentation (pH/DO gradients in reactors). Future directions:

Using CRISPR-Cas9 for precise multi-target gene editing in strains;

Optimizing fermentation parameters with AI algorithms to build real-time feedback control models, further increasing yield and reducing costs.