Fluorescence-Based Detection of Aflatoxin M1 in Milk using Immobilized Spores

Abstract

Spores of Bacillus megaterium were immobilized on a sensor disk by entrapment method followed by addition of pretreated milk and fluorogenic substrate, i.e., diacetate fluorescein with a response time of about 20 ± 5 min with ≥0.25 μg/L detection limit. Fluorescence was measured semiquantitatively using microbiological plate reader at specific excitation and emission spectra, i.e., 485 and 535 nm, respectively. High fluorescence within 20 ± 5 min is an indication of spore germination with the release of enzyme and its action on fluorogenic substrate. However, decrease in fluorescence indicates that milk is positive for AFM1 at 0.25 ppb. Reliability of fluorescence-based assay has been checked by spiking AFM1 in milk, which shows that fluorescence is inversely proportional to AFM1 concentration. The developed assay was evaluated with real food matrix such as raw, pasteurized and dried milk (n = 59), and it was compared with radioimmunoassay (Charm assay) and enzyme-linked immunosorbent assay which showed correlation of 0.94 and 1.0, respectively. Fluorogenic assay based on spore germination principle on sensor disk has the potential for routine monitoring of AFM1 in dairy farm, reception dock and manufacturing unit of dairy industry. Practical Applications Fluorescence-based detection of AFM1 in milk using immobilized spores on a sensor disk have a response time of 20 ± 5 min with a detection limit of ≥0.25 ppb. High fluorescence within 20 ± 5 min specifies germination of spores with the release of marker enzyme and its action on fluorogenic substrate, whereas decrease in fluorescence shows the presence of AFM1 in milk at 0.25 ppb. The assay was validated with radioimmunoassay (Charm assay) and enzyme-linked immunosorbent assay in real food matrix such as raw, pasteurized and dried milk. The assay has the potential for routine monitoring of AFM1 in dairy farm, reception dock and manufacturing unit of dairy industry.