Scalable anthocyanin extraction and purification methods for industrial application

Blackcurrant (Ribes nigrum L.) fruit is used in the food industry to produce various beverages, jams and other preparations. Waste blackcurrant skins from fruit pressing are obtained in large quantities from this sustainable and food-grade source. The material can be processed and extracted in large scale using acidified water, and subsequently purified using Solid Phase Extraction. The blackcurrant extracts are intensely coloured due to the presence of anthocyanins and have potential to act as natural colorants in consumer products. Blackcurrant extract was analysed using analytical HPLC, LC-MS, 1H NMR spectroscopy and total monomeric anthocyanin content assay (TMAC). Four major anthocyanins along with eight other polyphenols were identified in the crude extract; these were rutinosides and glucosides of cyanidin and delphinidin. Flavonols namely quercetin and myricetin were found in the aglycon form as well as mono- and di-substituted forms. Caffeic and p-coumaric acid were also present in the extract. Liquid-liquid extraction allows partitioning of specific metabolites based on their physical properties (neutral or charged species, aglycons, glucosides or rutinosides). It was observed that migration of different polyphenols out of the aqueous layer is possible by taking advantage of parameters such as the polarity of the organic solvent employed or pH of the aqueous layer. Use of ethyl acetate facilitates removal of neutral compounds, such as myricetin and quercetin (aglycons and glucosides) along with hydroxycinnamic acids (caffeic and p-coumaric acids), out of the aqueous extract. Isopropyl acetate selectively removes hydroxycinnamic acids, myricetin and quercetin, with the four anthocyanins (Dp-3-glc, Dp-3-rut, Cy-3-glc, Cy-3-rut) along with rutinosides of myricetin and quercetin remaining in the aqueous layer. Myricetin and quercetin rutinosides could be subsequently removed by extraction in ethyl acetate at higher pH (6.29), leaving a purified anthocyanin fraction. Preparative HPLC was utilised to isolate these polyphenols which were then fully characterised