Blue natural colorants for fermented foods

Danish title: Betydning af glucuronolering for stabiliteten af anthocyaniner

 

The overall practical aim of this project is to develop a blue anthocyanin-based colorant that is stable at low pH as encountered in fermented foods and dairy products. The approach taken is based on our joint skills within isolation, biosynthesis and enzymatic modification of anthocyanins and on molecular modelling of glycosyltransferases.
The color of anthocyanins strongly depends on the substituents present at the B-ring of the flavonoid sceleton. Increased hydroxylation and methoxylation at the 4´, 3´ and 5´ positions shifts the color from orange towards red and blue. The stability of the pigment may be improved by acylation of the sugar residue typically linked at the 3 position of the flavonoid skeleton by phenylpropanoids, or by formation of di-glycosides by addition of an additional sugar. In the present project, we want to exploit the possibility to form diglycosides in which one of the sugar residues carries a negative charge e.g. in form of a glucuronosyl residue. A soluble UGT enzyme (UDP-glucuronic acid: anthocyanin glucuronosyltransferase) able to carry out such a reaction has recently been characterized in flowers of red daisy (Bellis perennis) by Sawada et al. (J. Biol. Chem.) and shown to belong to the PSPGs (plant secondary product glucosyltransferases). The negative charge on the second sugar residue is thought to prevent nucleophilic attack and addition of water at the oxonium ion function of the chroman structure and thus mimics acylation by a phenylpropanoid.

At present, the pigment content of flowers of red daisy has been analyzed by HPLC analysis in combination LC-MS and NMR spectrometry. The pH stability of the glucuronosylated main pigment in comparison to the non-glucuronylated form of the pigment has been determined. Heterologous expression of the red daisy glucuronosyltransferase has been accomplished and homology modelling of the glucuronosyl transferase based on the molecular model of sorghum UGT85B1 and on available crystal structures are in progress with focus on residues affording binding of UDP-glucuronic acid and determining aglycon specificity. Site-directed mutagenesis to optimize glucuronylation of delphinidin based anthocyanins either by modification of the isolated glucuronosyl transferase or by modification of UGT85B1 is in progress.

The programme will serve to increase our understanding of the relationship between the molecular structure of anthocyanins and colour formation. It will also serve to increase our understanding at the functional level of additional members of the P450 and UGT superfamilies.

Functional assays of new anthocyanin pigments in food models are carried out at the Chr.Hansen Laboratories. Basic biochemistry and molecular biology at the Plant Biochemistry Laboratory

 

Researchers involved: Sarah Anne Osmani, Søren Bak, Birger Lindberg Møller

Foreign collaborators: Eric Jouenne, Chr. Hansen Laboratories; Anne Imberty
and Christelle Breton, Centre de Recherches sur les Macromolécules Végétales, CNRS, Grenoble, France