Screening for Antioxidant-Producing Actinomycetes

Antioxidants are essential for combating free radicals and preventing oxidative damage, making them imperative for our health and wellness. Actinomycetes are gram-positive bacterial species known for their filamentous growth and secondary metabolite production. This study aimed to identify the antioxidant-producing actinomycetes in our collection using a free radical assay. Briefly, radical scavenging was quantified using the color-changing reagent 2,2-diphenyl-1-picrylhydrazyl (DPPH). Isolates were grown in 2 L of A1 or A1DI media for marine or freshwater strains respectively made with starch, yeast extract, and peptone. Cultures were extracted using the polymeric XAD16-resin for 24 h, the resin was then rinsed with acetone to release secondary metabolites. The extract was dried and partitioned between ethyl acetate and water. The ethyl acetate partition was fractionated using silica gel SPE. For the assay, a 0.2 mM DPPH solution in methanol was prepared, and 109 fractions were tested in a 96-well microplate and compared against a negative control. Samples were tested in 8 serial dilutions from 512 μg/mL to 8 μg/mL to determine the EC50 of the antioxidant activity. After 30 min incubation, absorbance was measured at 515 nm to assess radical scavenging activity based on absorbance. Future directions are to grow the most active cultures in large-scale (6 L) to isolate the small molecules exhibiting free radical scavenging. This could lead to the discovery of novel antioxidant applications in healthcare, nutraceuticals, or beauty products supporting enduring wellness.