Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing.

The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.

Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Isatis tinctoria L. For the efficient production of flavonoids and evaluation of antioxidant activities.

In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 µg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 µg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC50 values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.