Nisin's development of biosensors

Biosensors based on Nisin leverage its specific antimicrobial activity or controllable gene expression properties, combined with signal transduction technologies such as fluorescence, bioluminescence, and electrochemistry, to enable rapid detection of spoilage bacteria or Nisin titer in food, thereby real-time evaluating food freshness. Various sensor types, including whole-cell and impedance-based sensors, have been developed for applications in milk, meat, and other food scenarios. Below is a detailed overview of their development and applications:

Main Types of Nisin Biosensors and Technical Principles

Fluorescent Whole-Cell Biosensors

These sensors are constructed using the Nisin-controlled gene expression (NICE) system. The core design involves linking reporter genes—such as red fluorescent protein (rfp) or anaerobic fluorescent protein (aFP)—to a Nisin-inducible promoter (Pnis) and transforming the construct into Lactococcus lactis. When the sensor encounters Nisin or Nisin-producing strains in food, the NisRK two-component system in L. lactis perceives the Nisin signal, activating the promoter to drive fluorescent protein expression. For example, a team from Shanghai Jiao Tong University developed two red fluorescent protein sensors that specifically respond to Nisin concentrations ranging from 2–200 ng/mL; a sensor incorporating mCherry fluorescent protein exhibits higher sensitivity, detecting Nisin as low as 1 ng/mL, suitable for milk and colonic simulation systems.

Bioluminescent Whole-Cell Biosensors

Modified bacterial luciferase operons (lux abcde) are linked to Nisin-inducible promoters via plasmids and transformed into strains like Lactococcus lactis NZ9800 or NZ9000. These strains’ NisRK genes sense Nisin and initiate luciferase gene transcription, producing bioluminescence without requiring exogenous substrates. This sensor boasts extremely fast response times, generating detectable signals within 10 minutes. It retains activity after lyophilization, with a detection limit of 0.1 pg/mL in pure solutions and 3 pg/mL in milk—sensitivity far exceeding traditional detection methods.

Impedimetric Electrochemical Biosensors

These sensors exploit Nisin’s antimicrobial property of disrupting bacterial cell membranes. Nisin molecules are immobilized on the surface of gold electrodes; when the sensor contacts food spoilage pathogens (e.g., Salmonella), interactions between Nisin and bacterial cell membranes alter the electrochemical impedance of the electrode surface. The sensor can selectively distinguish pathogenic from non-pathogenic Salmonella strains, with a detection limit as low as 1.5×10¹ CFU/mL. It is directly applicable for rapid detection of Salmonella in milk, reflecting the extent of pathogenic contamination and assessing food freshness.

Core Applications in Real-Time Food Freshness Monitoring

Dairy Preservation Monitoring

Dairy products like milk are prone to spoilage due to overgrowth of Salmonella and lactic acid bacteria. Both fluorescent and bioluminescent sensors are suitable for this 场景. For instance, fluorescent sensors can detect metabolites of Nisin-producing strains in milk, with 10 ng/mL Nisin sufficient to induce a fluorescent signal; bioluminescent sensors rapidly quantify residual Nisin or spoilage bacteria in milk, preventing dairy deterioration due to preservative failure or contamination.

Freshness Assessment of Meat and Aquatic Products

Protein-rich foods such as meat, fish, and shrimp are susceptible to spoilage by Salmonella and Pseudomonas. Impedimetric sensors monitor real-time freshness by detecting changes in the quantity of these pathogens. Unlike traditional colony counting methods, which take days, this sensor outputs impedance signals rapidly for on-site real-time monitoring, enabling early warning of meat spoilage.

Process Monitoring in Fermented Foods

In the production of fermented foods containing Nisin, sensors monitor Nisin synthesis by production strains to ensure stable preservative efficacy. For example, in fermented milk and fermented meat products, fluorescent sensors track Nisin concentration in real time, preventing shortened shelf life due to insufficient Nisin or flavor impairment from excessive addition.

Optimization Strategies and Application Advantages

Performance Optimization Methods

To address cytotoxicity from high Nisin concentrations, adaptive laboratory evolution can generate tolerant mutant strains, enhancing sensor stability in high-concentration targets. Precision cloning techniques like Golden-Gate cloning optimize vector construction efficiency and improve signal specificity. Additionally, lyophilization extends sensor storage life, making it suitable for on-site detection in food storage and logistics.

Core Application Advantages

Rapid Response: Fluorescent and bioluminescent sensors yield results within 10 minutes to several hours, far faster than traditional microbial culture methods.

Simple Operation: No complex pretreatment is required; some sensors directly detect live bacteria.

High Safety: Host strains of whole-cell sensors and Nisin itself meet food-grade standards, eliminating risks of chemical residues.

Broad Adaptability: By adjusting reporter genes or electrode materials, sensors can be adapted to detect various food types.

Current Challenges and Improvement Directions

Insufficient Scenario Adaptability

Some sensors exhibit reduced sensitivity in complex food matrices. For example, fluorescent sensors in colonic simulation systems require 50 ng/mL Nisin to trigger signals—far higher than the 10 ng/mL threshold in milk. Future efforts should optimize carrier structures to enhance anti-matrix interference capabilities.

Limited Large-Scale Application

Whole-cell sensors have strict storage requirements; although lyophilization improves this, long-term stability needs enhancement. Impedimetric sensors face high electrode production costs, necessitating the development of low-cost edible electrode materials.

Narrow Detection Range

Most current sensors focus on Nisin or Salmonella detection. Future integration of multi-reporter genes or composite biorecognition elements could enable simultaneous detection of multiple spoilage bacteria and toxins, further improving food freshness monitoring systems.