Browsing by Subject "Fluorescent assay"

Lactobacilli especially Limosilactobacillus reuteri’s strains inhabit the GI tract of humans with glycerol/diol dehydratase activity metabolizing glycerol and producing a broad-spectrum antimicrobial system called reuterin. It consists of 3-hydroxypropanal (3-HPA), acrolein, and its derivatives. Due to the toxic activity of ubiquitous acrolein, an analytical toolbox to determine acrolein formation by food cultures is needed. We developed assays to estimate microbial formation of acrolein using a colorimetric method based on tryptophan and a fluorescence-based approach with 2-amino-1-methyl benzimidazole (AMBI) as a probe. We compared tryptophan and AMBI-based quantification of reuterin produced by resting cells or during the growth of Lb. reuteri DSM 20016. With 600 mM glycerol, resting cells produced 329 ± 35 mM 3-HPA as quantified by HPLC-RI, and 390 ± 13 mM of 3-HPA/acrolein based on the colorimetric method with 3-HPA as standard. Acrolein (40 ± 11 mM) was detected using an AMBI probe. We also detected 3-HPA and acrolein formation during the exponential growth phase in the presence of 50 mM glycerol in different media. Also, as acrolein induces redox stress, redox potential and sensitivity to reuterin/acrolein of the engineered green fluorescence protein (roGFP2) were determined. Ultimately, the roGFP2 gene was tried to clone in E. coli (JM109) using the pTH1mp constitutive expression vector to establish as a biosensor for acrolein detection. Our results suggest that quantification of acrolein by fluorescence-based approaches and biosensors constitute novel methods to estimate any risk of acrolein formation in presence of glycerol/diol dehydratase-positive microbes and glycerol during food fermentation.