A novel agar diffusion assay for qualitative and quantitative estimation of ε-polylysine in fermentation broths and foods

A novel agar diffusion assay was developed and validated for the analysis of ε-polylysine (ε-PL) in fermentation broths and foods. The basis for the method is the strong electrostatic interaction between the positively charged ε-PL and methylene blue dye that causes the appearance of a clear diffusion zone of ε-PL on an agar plate containing methylene blue. A highly linear relationship between the diffusion zone diameter and the log ₁₀ ε-PL concentration was found. Interfering substances were also discovered in the process of estimating of ε-PL, but their effects could be compensated or eliminated by properly preparing ε-PL standards or diluting the unknown ε-PL sample. With this method, ε-PL concentrations ranging from 0.2g/l to 8g/l in fermentation broth could be detected reliably with an assay time about 5h. In addition, this method also allowed detecting as low as 2mg/kg ε-PL of orange-juice, 10mg/kg ε-PL of cake and of pork-ham. To the best of our knowledge, this is the first time to apply electrostatic interaction between the target substance and charged dyes on agar plate in an analytical assay. This proposed agar diffusion assay can be used in ε-PL-production and food industry as well as in other basic researches because of its simplicity, cost-effectiveness and high specificity. Moreover, the method described here offers a new research area for the determination of polycationic and polyanionic substances.