Sustainable endospore-based microreactor system for antioxidant capacity assay.

Abstract

A novel endospore-based microbial method for "post-additional" antioxidant capacity assay was developed. The technique was based on oxidation and catalysis of the 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) by Bacillus subtilis 168 endospores in the presence of dissolved oxygen. Coat protein A (CotA), which belongs to the endospore coat, was expressed, purified, and assessed for its ability to oxidize ABTS into the ABTS(•+) radical cation. The wild-type endospore necessary for oxidizing ABTS into ABTS(•+) radical cation was confirmed by knocking out the cotA gene from B. subtilis 168 by homologous double exchange. Findings revealed that the catalytic activity of the endospores may be attributed to the presence of the CotA protein. The use of endospores instead of purified enzymes to prepare ABTS(•+) greatly reduced the assay cost and eliminated the need to purify and store of enzymes. The self-life of the radical cation was kept stable for at least 12 days without addition of a stabilizer and laccase inhibitor. This behavior enables the large-scale preparation of ABTS(•+). The antioxidant capacities of the individual antioxidants and fruit samples were easily quantified and compared using the proposed method. The developed technique can be further developed as a high-throughput screening technique for antioxidants.