The residue limit standard of Nisin

Nisin, a naturally occurring bioactive antimicrobial peptide, is widely used in food preservation and other fields. The following is information regarding its residue limits and detection methods:

Residue Limit Standards

Currently, residue limits for Nisin in food vary across countries and regions, and relevant standards may be updated with regulatory revisions. Internationally, the Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) has not set specific residue limits for Nisin, as it is considered a safe food preservative with no need to specify an acceptable daily intake (ADI). However, in practical applications, food manufacturers must comply with national regulations. For example, in some countries, Nisin is permitted in dairy products and other foods, with residues required to be controlled within reasonable ranges—typically no more than several hundred mg/kg, with specific values varying by food type.

The U.S. Food and Drug Administration (FDA) has classified Nisin as a Generally Recognized as Safe (GRAS) substance, allowing its safe use under conditions consistent with current Good Manufacturing Practices (GMP) without specific residue limits.

In China, Nisin is approved for use in various foods, such as dairy products and prefabricated meat products. Specific residue limits are determined based on food categories in accordance with relevant food additive standards, generally ranging from several tens to several hundred mg/kg.

Detection Methods

Agar Diffusion Method: This is the most commonly used quantitative bioassay for Nisin. Its principle is to visualize the bacteriostatic effect through the growth of indicator bacteria on an agar surface. By diffusing Nisin standard solutions and unknown samples on the same plate, the titer is calculated using the diameter of the inhibition zone and standard titer. Common indicator strains include Micrococcus lysodeikticus, Staphylococcus aureus, and Micrococcus luteus. To promote Nisin diffusion in agar, Tween is often added to the detection medium. This method is simple, convenient, and economical but is susceptible to interference from other substances, potentially leading to false positive results.

Immunoassays: Leveraging the rapidity, directness, and sensitivity of immunological methods, quantitative detection techniques for Nisin have been developed, such as enzyme-linked immunosorbent assay (ELISA). Studies have reported that an ELISA method using polyclonal antibodies against Nisin Z can detect limits as low as 0.23 nmol/L in buffer, 0.5 nmol/L in milk, and 1.49 nmol/L in complex media. This method is highly sensitive but may produce unreliable results due to cross-reactivity with structurally related compounds.

High-Performance Liquid Chromatography (HPLC): HPLC enables accurate qualitative and quantitative determination of Nisin with high precision, a minimum detection limit of 10 IU/mL, and reliable analysis of both solid and liquid samples. It is relatively simple to operate, easy to popularize, and suitable for Nisin content detection in production enterprises and testing institutions.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): This method offers high sensitivity and accuracy. Studies using LC-MS/MS to determine Nisin A and Nisin Z in milk have reported limits of quantification of approximately 12.9 µg/kg and 10.9 µg/kg, respectively, with high recovery rates and low coefficients of variation, making it suitable for detecting Nisin in complex matrices like milk.

Other Methods: These include turbidimetry, ATP bioluminescence assay, green fluorescent protein (GFP) assay, and micellar electrokinetic chromatography. For example, a detection method based on regulatory proteins encoded by Lactococcus lactis and GFP variant genes can detect Nisin in milk as low as 0.2 ng/mL. This method is highly sensitive and can avoid interference from potential inhibitory substances in samples through extensive dilution.