Microplate bioassay for nisin in foods, based on nisin-induced green fluorescent protein fluorescence.
A plasmid coding for the nisin two-component regulatory proteins, NisK and NisR, was constructed; in this plasmid a gfp gene (encoding the green fluorescent protein) was placed under control of the nisin-inducible nisF promoter. The plasmid was transformed into non-nisin-producing Lactococcus lactis strain MG1614. The new strain could sense extracellular nisin and transduce it to green fluorescent protein fluorescence. The amount of fluorescence was dependent on the nisin concentration, and it could be measured easily. By using this strain, an assay for quantification of nisin was developed. With this method it was possible to measure as little as 2.5 ng of pure nisin per ml in culture supernatant, 45 ng of nisin per ml in milk, 0.9 microg of nisin in cheese, and 1 microg of nisin per ml in salad dressings.
Reunanen
J
J
Applied Chemistry and Microbiology, University of Helsinki, FIN-00014 Helsinki, Finland.
Saris
P E J
PE
D000818:Animals; D000900:Anti-Bacterial Agents / Q000032:analysis*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods*; D002611:Cheese / Q000032:analysis; D005453:Fluorescence; D005520:Food Preservatives / Q000032:analysis*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008892:Milk / Q000737:chemistry; D009561:Nisin / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005520:Food Preservatives; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D009561:Nisin; D049452:Green Fluorescent Proteins