Final Report Summary - MICROSMETICS (Exploitation of microbial biodiversity for the discovery and development of novel cosmeceutical agents)
The aim of MICROSMETICS project was to discover and carry to the stage of development innovative products in the area of cosmeceuticals originating from global biodiversity using emerging and state of the art technologies in the field of biotechnology, natural products chemistry and applied microbiology.
More specifically, MICROSMETICS scientific concept involved the discovery of novel natural products originating from global microbial biodiversity. Already existing culture collections were exploited incorporating modern high throughput platforms (in silico & in vitro) for the rational and targeted selection of the most promising strains. Advanced analytical approaches and techniques have been applied for the efficient, accelerated and advantageous isolation and identification of natural constituents as well as the quality assessment of the lead products. A broad spectrum of bioassays and novel analytical approaches were incorporated for the evaluation of anti-ageing, more specifically anti-oxidant, skin-protecting, and skin-whitening activity of all derived products. Attention was given to the selection and optimisation of fermentation technologies used for the production of final lead products to ensure sustainability.
In order to achieve those goals, as a first step a Rational Drug Design approach had been developed. Starting from the CosIng Repository of the European Commission, an accurate functional prediction model was created for all known cosmetic functions. Additionally it was constructed the homology models of specific cosmetic target receptors (tyrosinase, elastase, hyaluronidase and collagenase) for which the appropriate in vitro tests have already been developed. More than 40.000 known microbial metabolites were processed through a consensus scoring prediction protocol using: a) functional prediction model b) virtual screening procedure for the above 4 selected receptors c) similarity search based on all known molecules from literature that bind to the specific receptors and d) toxicological profile filtering. Combining all the results with functional prediction model and toxicological profile filtering, 100 microorgnanisms have be selected that can produce those metabolites or analogues.
Among them 54 fungi and 56 actinomycetes from the global biodiversity (including strains from Antarctica, Alaska, Spain, New Caledonia, Hawaii, South Africa, Comoros Island etc) were cultivated under “nutritional arrays” (different culture conditions) in order each single strain to produce ten different extracts and thus exploit all the potential chemodiversity that microorganisms can produce. In total 1082 sample extracts (614 actinomycetous extracts & 425 fungal extracts) have been generated, then were filtered and prepared in 96 barcoded well format and were shipped for high throughput screening.
Among the 1082 extracts the top 100 most bioactive extracts were selected for metabolomics analysis and a strategy combining UHPLC/Orbitrap-HRMS, in positive and negative modes, with multivariate statistical methods was applied. All derived chromatograms have been analyzed and a positive correlation between the profiles of the extracts with the aforementioned bioassays was observed. Thus, through this metabolomics analysis the 20 most promising extracts (19 strains under 14 conditions) that represent the clusters have been forwarded for large-scale cultivation and for confirmation of the activity. In total 8 fungi and 12 actinomycetes were selected for further investigation - 5 with antioxidant activity, 5 with skin whitening, 8 with skin protecting activity, 1 combining antioxidant and skin protecting activity and 1 combining skin whitening and skin protecting activity.
All the selected strains have been forwarded for large-scale cultivation and bioassay-guided isolation of novel molecules. Biomass production though fermentations and production metabolites were checked in parallel in order to assure the same chemical profile of the extracts. Thus, several cultivation parameters were adjusted. Biomass was extracted using different protocols and extracts went to further chemical investigation. Different chromatographic methods were used to fractionate the most active extracts of the selected strains, which were also tested for their biological activity and novel bioactive compounds were isolated.
After taking into consideration the results from the complete set of biological in vitro and cell-based experiments, cytotoxicity and physicochemical characteristics the two most promising candidates were transferred for scale-up production in bioreactors: one fungi - the most promising for antiageing activity and one actinomnycete- the most promising for skin whitening activity. The produced extracts were re-evaluated for their activity in cell free and cell based assays and have confirmed their potential.
Finally both extracts produced in bioreactors have been forwarded to be incorporated in final cosmetic products and their properties including physicochemical and efficacy have been evaluated. Furthermore in order to evaluate the potential for commercializing those products a first feasibility study has been performed.
Overall we consider MICROSMETICS as a successful model of an Industry – Academia collaboration, as through the secondments that were implemented there was a significant transfer of know how between the partners that were involved in the project and all researchers have expanded their expertise, their skills and overall their professional potentials. Also the establishment of this network has clearly accelerated the research capacity for all the involved academic and industrial partners.
More specifically, MICROSMETICS scientific concept involved the discovery of novel natural products originating from global microbial biodiversity. Already existing culture collections were exploited incorporating modern high throughput platforms (in silico & in vitro) for the rational and targeted selection of the most promising strains. Advanced analytical approaches and techniques have been applied for the efficient, accelerated and advantageous isolation and identification of natural constituents as well as the quality assessment of the lead products. A broad spectrum of bioassays and novel analytical approaches were incorporated for the evaluation of anti-ageing, more specifically anti-oxidant, skin-protecting, and skin-whitening activity of all derived products. Attention was given to the selection and optimisation of fermentation technologies used for the production of final lead products to ensure sustainability.
In order to achieve those goals, as a first step a Rational Drug Design approach had been developed. Starting from the CosIng Repository of the European Commission, an accurate functional prediction model was created for all known cosmetic functions. Additionally it was constructed the homology models of specific cosmetic target receptors (tyrosinase, elastase, hyaluronidase and collagenase) for which the appropriate in vitro tests have already been developed. More than 40.000 known microbial metabolites were processed through a consensus scoring prediction protocol using: a) functional prediction model b) virtual screening procedure for the above 4 selected receptors c) similarity search based on all known molecules from literature that bind to the specific receptors and d) toxicological profile filtering. Combining all the results with functional prediction model and toxicological profile filtering, 100 microorgnanisms have be selected that can produce those metabolites or analogues.
Among them 54 fungi and 56 actinomycetes from the global biodiversity (including strains from Antarctica, Alaska, Spain, New Caledonia, Hawaii, South Africa, Comoros Island etc) were cultivated under “nutritional arrays” (different culture conditions) in order each single strain to produce ten different extracts and thus exploit all the potential chemodiversity that microorganisms can produce. In total 1082 sample extracts (614 actinomycetous extracts & 425 fungal extracts) have been generated, then were filtered and prepared in 96 barcoded well format and were shipped for high throughput screening.
Among the 1082 extracts the top 100 most bioactive extracts were selected for metabolomics analysis and a strategy combining UHPLC/Orbitrap-HRMS, in positive and negative modes, with multivariate statistical methods was applied. All derived chromatograms have been analyzed and a positive correlation between the profiles of the extracts with the aforementioned bioassays was observed. Thus, through this metabolomics analysis the 20 most promising extracts (19 strains under 14 conditions) that represent the clusters have been forwarded for large-scale cultivation and for confirmation of the activity. In total 8 fungi and 12 actinomycetes were selected for further investigation - 5 with antioxidant activity, 5 with skin whitening, 8 with skin protecting activity, 1 combining antioxidant and skin protecting activity and 1 combining skin whitening and skin protecting activity.
All the selected strains have been forwarded for large-scale cultivation and bioassay-guided isolation of novel molecules. Biomass production though fermentations and production metabolites were checked in parallel in order to assure the same chemical profile of the extracts. Thus, several cultivation parameters were adjusted. Biomass was extracted using different protocols and extracts went to further chemical investigation. Different chromatographic methods were used to fractionate the most active extracts of the selected strains, which were also tested for their biological activity and novel bioactive compounds were isolated.
After taking into consideration the results from the complete set of biological in vitro and cell-based experiments, cytotoxicity and physicochemical characteristics the two most promising candidates were transferred for scale-up production in bioreactors: one fungi - the most promising for antiageing activity and one actinomnycete- the most promising for skin whitening activity. The produced extracts were re-evaluated for their activity in cell free and cell based assays and have confirmed their potential.
Finally both extracts produced in bioreactors have been forwarded to be incorporated in final cosmetic products and their properties including physicochemical and efficacy have been evaluated. Furthermore in order to evaluate the potential for commercializing those products a first feasibility study has been performed.
Overall we consider MICROSMETICS as a successful model of an Industry – Academia collaboration, as through the secondments that were implemented there was a significant transfer of know how between the partners that were involved in the project and all researchers have expanded their expertise, their skills and overall their professional potentials. Also the establishment of this network has clearly accelerated the research capacity for all the involved academic and industrial partners.