In the frame of MICROSMETICS EU project 56 potential candidate actinobacteria strains of global biodiversity were selected to be studied using OSMAC strategy. In total 614 extracts were produced and cell-free bioassays have been used for the evaluation of their skin-whitening bioactivity.
Among the initial 614 extracts, the actinomycete strain CA-129531 of the genus Streptomyces, originated from Maritinique and cultivated in the fermentation medium DNPM, exhibited the most promising skin-whitening activity (i.e. tyrosinase inhibition). This activity was confirmed in cell-based assays in mouse melanocytes (B16F10 cell line). Preliminary study of this strain, including the scale up fermentation in 1lt and bioguided fractionation of the active EtOAc extract, led to the isolation and identification of trichostatin A and trichostatic acid.
Scale-up process optimization was performed in a bioreactor Biostat C+ (total volume 30 kg). Direct liquid/liquid extraction of the culture medium with EtOAc was performed and the yield of trichostatin A was measured in different experiments performed under modified media. The highest amount of this marker was observed when using media DNPM*3, a modified formulation of DNPM that used as carbon source dextrose in agreement with requirements of cosmetic legislation.
After confirming the tyrosinase inhibition in cell-free assay of the EtOAc extract of this broth (IC50=63.27μg/ml), preparative HPLC was used in order to bioguided isolate the active compounds. The full set of spectroscopic data (HRMS and NMR) was recorded for all active isolated compounds in order to unambiguously elucidate their structure, while it was also identified the main metabolite responsible for this activity (IC50=3.07μg/ml).
Therefore, this extract can be considered as potential candidate for industrial development and this optimized small-scale process can then be transferred to pilot scale following established scale-up strategies in cosmeceutical industry.