Tetrazolium-Based Rapid Colorimetric Assay to Determine Bacteriocin Activity

Abstract

The aim of this study is to develop a simple, rapid, and accurate method for quantitative determination of bacteriocin activity. The method involves the use of 2,3,5-triphenyltetrazolium chloride (TTC), which is a colorless water-soluble salt. Only viable cells take up the compound and reduce it intracellularly to its red-colored and water-insoluble formazan dye. Bacteriocin, nisin and lacidin-A were assayed using the sensitive indicator Lactobacillus delbrueckii subsp. lactis ATCC 4797 whereas pediocin PO2 was assayed against Lactobacillus bulgaricus OSU 135. The major factors affecting the reduction of TTC, such as the reagent concentration, incubation period, temperature, and pH were adjusted so that optimal reduction of TTC by the indicator microorganisms could be achieved. Two-fold dilutions of the bacteriocins were mixed with a standardized indicator culture and incubated for 30 min. Then 0.2% TTC was added and the mixture (pH 6.0) was incubated at 37 oC for 20 min. After the incubation, formazan was extracted from the cells with methanol and the absorbance was measured at 485 nm. The amount of formazan formed by the survivor cells was compared with survivor counts and zone of inhibition method. The dose-response plot for the TTC-based bacteriocin assay was linear over a wide range bacteriocin concentration. A high correlation ( R2 > 0.95 ) was between viable cell count and TTC reduction for three of the bacteriocins tested. The new assay can be completed in one hour, compared to one or two days with microbiological assays. Overall, the procedure is simple and easy to carry out.

Keywords

• Bacteriocin; 2
• 3
• 5-triphenyltetrazolium chloride (TTC); TTC-based bacteriocin assay
• Nisin
• Lactacin A

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