Red clover isoflavonoids as anthocyanin color enhancing agents in muscadine wine and juice

Isoflavonoid extracts from red clover (Trifolium pratense) leaves were found to enhance overall color and stability of anthocyanin 3,5-diglucosides present in muscadine grape (Vitis rotundifolia) juice and wine through intermolecular copigmentation reactions. Predominant isoflavonoids present in red clover included formononetin, biochanin A, and prunetin and were the major polyphenolics identified to influence anthocyanin color and stability. Since red clover isoflavonoids have poor water solubility characteristics, this allowed for removal of extraneous non-isoflavonoid compounds using hot water and subsequent extraction with ethanol. Isoflavonoid solubility was evaluated as a function of ethanol concentration with recoveries up to 57% found in 20% solutions. Changes in maximum absorbance, total soluble phenolics, isoflavonoids, and anthocyanins were evaluated in muscadine juice and wine following the addition of isoflavonoid extracts with maximum color enhancement found at an anthocyanin to cofactor ratio of 1:8, after which their solubility was prohibitive. Additionally, dried leaves and ethanolic extracts of red clover were added prior to and following fermentation of muscadine wine (11% ethanol) stimulating the natural copigmentation that takes place during red wine fermentation and aging processes. Once fermentation was complete, finished wines were evaluated over a 9-week storage period at 20 and 37 °C. Despite low levels of isoflavonoids present, color improvement and anthocyanin stability was observed in the wines during storage. Little information is available on copigmentation reactions occurring in actual food systems, yet red clover isoflavonoids proved to be novel and effective color enhancing compounds when used in low concentrations in young muscadine wines.