13 ERA IB: ANTHOcyanin production PLatform Using Suspension cultures (ANTHOPLUS)

Anthocyanins are water-soluble pigments that colour the fruit and flowers of many plants. More than 635 different anthocyanins have been identified, distinguished by methylation, glycosylation and acylation with both aliphatic and aromatic groups. There is mounting evidence that consumption of anthocyanin-rich food promotes health, supported by many recent studies of anthocyanin-rich fruits such as blueberry, bilberry and cranberry.
Their relative abundance in the diet and their potency against a range of chronic diseases have made anthocyanins the subject of intense research in experimental and preventive medicine and, more recently for formulating natural colours. However, the limited range of anthocyanins commercially available and the expense of pure preparations mean that most research is done with crude extracts of plants which are not standardised with respect to the particular anthocyanins they contain, nor the amounts of each anthocyanin in the extract. Variations in anthocyanin decoration account for differences in colour stability and hue of anthocyanins and underpin the need for developing production systems for pure anthocyanins for investigating the effects of chemical specificity on uptake, signalling and physiology, toxicity of anthocyanins for medical applications and for developing new formulations in the natural colours industries.

In ANTHOPLUS novel plant cell cultures will be developed for the stable production of a wide variety of anthocyanins. The novel procedure for generating these cultures means that they are not constrained by prior art. Our cell cultures, uniquely, allow sustained, high level production of diverse anthocyanins. Enhanced supplies of pure anthocyanins will be highly valuable for the colourants industry to investigate the effects of decorations, co-pigments, pH on colour and stability to provide a robust scientific foundation for developing new plant sources of natural colourants and new formulations for natural colours.

We aim to develop methods for large scale production of pure anthocyanins, of varying complexity in side chain decoration, or labelled with stable isotopes from plant cells.

A robust plant tissue culture system will be used to produce large amounts of anthocyanins in a process spanning the whole value-chain from synthesis in novel suspension cultures, scale-up, preparative purification, assessment of bioactivities to distribution for sales. Our unique cell lines, scale-up and purification procedures will offer reliable commercial production, for the first time.

We will develop methods for enhanced production, with easier extraction, by producing cultures that excrete anthocyanins, and for production of highly decorated anthocyanins using new plant sources for cell cultures. A major component of this project will be scale up activities for culturing plant cells for specialty anthocyanin production in small industrial scale fermenters. Besides preparative extraction methods, we will develop methods for the production of purified 13C-labelled anthocyanins for research on their absorption (bioavailability) and in vivo metabolism (pre- and post-absorption) in humans and animals. A fine chemicals company will assay the quality/purity /stability and bioactivity of anthocyanins compared to those from other sources.

The exponential increase in interest in dietary anthocyanins means there is already a significant market for pure anthocyanins and stable isotope labelled anthocyanins amongst medical researchers. There is significant interest in pure anthocyanins from the natural colorants sector.
Pure anthocyanins will be brought to market by an SME who will commission specialty anthocyanins, develop plant-specific ANTHO-kits and expand their product catalogue. We plan to expand the EU natural colours market with new trial products through a market-led business development strategy, backed up by a quality technical data package and customer demonstrations.

Even though science has identified more than 600 different anthocyanins, only a few are commercially available in a pure state, and these are very expensive. The commercial interest in natural colorants is increasing, even though challenges exist. One challenge is that natural colorants do not provide the same range of hues as are available with synthetic colourants. For example, anthocyanin pigments, derived from fruit or vegetables, exhibit reversible structural changes, when the pH of the environment changes. These cause a hue change from red to purple to blue, as the food matrix changes from acidic to basic. This characteristic can create issues for formulators wishing to use anthocyanins in products. To find stable anthocyanins that can overcome this problem is one of the major tasks of ANTHOPLUS.

In terms of levels of production, ANTHOPLUS cannot and does not intend to provide anthocyanins in amounts that could be used directly as natural food colourants. The margins for natural colours are very small; they are derived largely from extraction of waste products of grape skins from the wine industry, and once extracted the source material is reused for oil extraction. However, the cell cultures developed through ANTHOPLUS will provide reliable production systems for anthocyanins as fine chemicals. These products will be of high purity and developed at relatively low cost using the ANTHOPLUS customised, sustainable technology platform. Such fine chemicals will greatly facilitate analysis of foods, and human/animal tissues by providing high quality standards. They will also facilitate metabolomic analysis of plant tissues by providing standards for HPLC and LC-MS analyses. Such analysis will be further facilitated by offering kits for analysis of specific crops. They can and will also be used to investigate the impact/performance of anthocyanins as natural colourants on the lab scale and in pilot production of the respective natural colourants. However, production will not exceed this level during this project.

From our collective experience as collaborators in research into the health-promoting properties of diets rich in anthocyanins, there is a pressing need, within the medical and nutritional research communities, for a source of purified, high quality anthocyanins with which to conduct cellular assays to assess mechanisms of action, to serve as dietary supplements and to provide standards for the compositional analysis of different foods and to assay bioavailability and metabolism. Many of the researchers interested in such a source are already clients of P4 (PMC). Therefore one of the first routes for exploitation of the outcomes of the ANTHOPLUS project will be the existing client base and newsletter list (more than 2,500 contacts) of P4 (PMC) and the PlantMetaChem/TransMIT newsletter. Other means of dissemination will be through advertisements in Natural Products journals and journals focussed on nutritional research, and presence and presentation of new products at related conferences and by scientific dissemination by customers.