ANTHOcyanin production PLatform Using Suspension cultures

Anthocyanins, pigments found in plants, have enormous potential as natural food colorants offering a range of colours from orange through red and purple to blue. Public concern over the toxicological safety of synthetic food colorants is increasing, resulting in the consequent banning of several red food dyes. There has been rising interest in the development and application of anthocyanins as food colorants. Most anthocyanins (nearly 700 have been described in nature) are not available commercially. One of the major problems with the widespread adoption of anthocyanins as natural colorants is the difficulty of reproducing the intense blue colours achieved in nature (cornflower, gentian, delphinium) in natural colour formulations. There is considerable interest in achieving strong blue anthocyanin preparations and to improve the stability of anthocyanins such that they can be used as industrially-reliable, natural colorants replacing synthetic ones, like Synthetic Blue Number One. Promising blue colours based on anthocyanins from plant cell cultures were recently established within the AnthoPLUS (ANTHOcyanin production PLatform Using Suspension cultures) ERA-IB project (2014-2017). AnthoPLUS produces anthocyanins, of varying complexity in side chain decoration, or labelled with stable isotopes as fine chemicals. These can be used for assaying the composition of feedstock for natural colours, for bioavailability, bioefficacy and mechanistic research in experimental medicine and as standards for assaying natural colorant extracts for improved formulations. The purified anthocyanins are also of high interest to the natural colours industry for pilot analysis of the stability, response to pH, light, heat, interactions with other compounds and processing and colour spectrum of new natural colours. The production of new anthocyanins from plants spans the whole of the value-chain from synthesis in novel suspension cultures, scale-up, preparative purification, to distribution for sales. The consortium of the AnthoPLUS project generated a number of novel plant cell cultures for production of various anthocyanins at high levels. In our studies, we gained new insights on the accumulation and transport of anthocyanins. Notably, a transporting signal has been identified which might enable the secretion of anthocyanins into the cultivation medium in the near future. To obtain anthocyanins labelled with stable isotopes for biomedical research particular growth protocols for plant cell cultures were developed. The stable growth of the plant cell cultures has been scaled-up from flask to bioreactors allowing for further transfer on industrial production scale. For the isolation of various anthocyanins as fine chemicals an automated two-step purification process has been established. Additionally, methods for analyzing the purity and stability of the preparations have been developed and applied. Cell models and assays for bioactivity profiling (cyto- and genotoxic, as well as antioxidant, anti-carcinogenic and anti-inflammatory properties) of anthocyanins were established and applied on a number of isolated compounds from AnthoPLUS and compared with the effects of reference compounds and degradation products. First commissions of pure anthocyanins have been realized by the involved SME reflecting the commercial interest in our products. This success has clearly been supported by the active promotion of the project and related products on conferences and fairs as well as by the established website (www.anthoplus.com) and the link to a commercial platform (http://www.plantmetachem.com/). AnthoPLUS has been generously supported by the ERA-NET Industrial Biotechnology 4th Joint Call, funded by the respective national and regional agencies. The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK, Germany, coordination), John Innes Centre (JIC, UK), Norwegian University of Life Sciences (NMBU, Norway), Institute of Food Chemistry and Toxicology of the University of Vienna (UNIVIE, Austria, sub-contractor) and the TransMIT GmbH, Project Division PlantMetaChem (PMC, Germany), were partners within the AnthoPLUS consortium. Without the whole Consortium Team, these results would not have been possible to see through.

ANTHOPLUS will produce anthocyanins in a robust new tissue culture system that develops the potential of plant cells as green factories for the stable production of natural plant products. Objectives: Our aim is to produce purified anthocyanins, of novel complexity in side chain decoration, or labelled with stable isotopes for assaying the composition of feedstocks for natural colours, for bioavailability, bioefficacy and mechanistic research in experimental medicine and as standards for assaying natural colorant extracts for improved formulations. Production of these compounds will span the whole value-chain from synthesis, scale-up, preparative purification, assessment of toxicity and bioactivities to distribution for sales. Expected results: A diversit y of anthocyanins in purified form will pave the way for new products and formulations in the food, pharma and cosmetics industries. By integrating the steps in the production pipeline, purified anthocyanins will be available to researchers and industry t ogether with bioactivity, bioavailability and biotoxicity data by the completion of the project. We will enhance production using cultures that excrete anthocyanins and produce highly decorated anthocyanins,using new plant sources for cultures. Highly dec orated anthocyanins will provide novel fine chemicals for testing stability, colour range, anti-oxidant and co-pigmentation properties for new natural colours and formulations. Exploitation: Demand for the products of ANTHOPLUS will be from clients within the bioanalytical, pharmaceutical and food industries. These new products will be produced via a market-led business development strategy, backed up by a quality technical data package and customer demonstrations. Successful commercialization will be ens ured by (i) the novelty and sustainability of the production approach (P2 (JIC) has IP on the culture production system), and (ii) the outstanding expertise of P4 (PMC) in the marketing of natural compounds.