As natural colorants get more attention in the food industry, efforts on anthocyanin (ACN) stabilization and color expression have increased for their incorporation in food products. Studies show enhancement of ACN color performance and resistance to degradation by stabilizing the pigment via intramolecular copigmentation. This reaction occurs between the ACN chromophore and its covalently bound acyl group on the glycoside. In plants, most acylating groups exist in the trans-isomeric configuration but can undergo excitation under ultraviolet and visible light to induce the cis-conformation. The applied radiant energy affects the isomerization barrier, causing the molecule to adopt its excited state, and producing a molecule with different chemical characteristics. Photochromism, defined as “light-induced, reversible change in color,” is the reaction that occurs when photoisomerization of molecules lead to a change in color. Relatedly, cis- and trans- acylated ACN have been known to exhibit differences in color expression and stability, but details on the factors affecting photochromism has not been well studied. The overall objective of this study was to investigate the conditions that influence photochromism of acylated ACN and compare the cis-trans isomers’ spectroscopic characteristics, colorimetry, and stability in various pHs.
In the first objective, the effects of irradiation time and excitation energy on ACN cis-trans isomerization and color expression were studied. East Asian eggplants were chosen as the source of the pigment, due to their simple ACN profile that contained the trans-isomer necessary to induce isomerization. Delphidin-3-(trans-p-coumaroyl)-rutinoside-5- glucoside, delphinidin-3-(cis-p-coumaroyl)-rutinoside-5-glucosde, and a semi-crude extract containing both isomers were characterized, standardized, and subjected to excitation (UV chamber at 254 nm, 365 nm, visible light with D65 lamp, and F2 lamp) for up to 20 hours. All four radiant energies induced photoisomerization of trans-to-cis and cis-to-trans photoisomerization, but to varying extents, equilibria, and under different exposure times. Visible energy induced greater trans-to-cis isomerization while UV induced greater of the reverse reaction. Cis-acylated delphinidin showed a more saturated color and stability in pH 1 compared to its trans-counterpart and exhibited a greener hue (h*ab 130 deg) in pH 8, compared to a bluer hue of the trans-isomer (h*ab 188 deg).
The second objective explored differences in photoisomerization reactivity of mono- and di-acylated ACN with varying hydroxycinnamic acylation patterns. For this objective, red cabbage was selected for its ACN profile with high abundance of varying mono- and di-acylated compounds. Three monoacylated and three diacylated derivatives of cyanidin-3- sophoroside-5-glucoside (Cy-3-soph-5-glu) with different hydroxycinnamic acids— p-coumaric acid (pC), ferulic acid (fer), and sinapic acid (sin)— all in the trans-configuration – were isolated and reconstituted in acidified methanol, then subjected with visible light (F2 lamps) for 20 hours. Pigment isomerization was monitored and quantified with uHPLC-PDA-ESI-MS/MS. Greater extent of photoconversion was observed for mono- acylated pigments than di-acylated pigments. Cy-3-trans-pC-soph-5-glu produced the greatest amount of the cis-isomer at 53.5% total peak area under the curve (at 510 – 540 nm under LCMS), followed by Cy-3-trans-sin-soph-5-glu (47.1%), then Cy-3-trans-fer- soph-5-glu (40.0%). When comparing among the diacylated pigments, Cy-3-trans-sin-sin- soph-5-glu produced the greatest amount of the cis-conformation at 45.6% AUC, in comparison to Cy-3-cis-sin-fer-soph-5-glu and Cy-3-cis-pC-sin-soph-5-glu that produced less than 30% of the cis-isomer. Lastly, regioisomers of Cy showed contrasting behavior. Acylation at the C6 glycosidic position isomerized extensively (47.1%), but not at the C2 glycosidic position (0%).
The third objective was to evaluate the cis-, trans-, and a mixture of the two isomers’ spectroscopic characteristics, color expressions, and degradation kinetics at a pH range that’s relevant for the food industry. The sinapic acid family—containing Cy-3-(2’’-trans)- sin-soph-5-glu, Cy-3-(6’’-trans-sin)-soph-5-glu, Cy-3-(6-trans-sin)-(2-trans-sin)-soph-5- glu were extracted from red cabbage extract. These trans-acylated ACNs were irradiated to produce two mixtures of cis-trans acylated ACNs for both mono- and di-acylated Cy. The mixtures were further isolated to produce two cis-acylated ACNs: Cy-3-(6’-cis-sin)- soph-5-glu and Cy-3-(6’-cis-sin)-(2’-trans-sin)-soph-5-glu. Spectrophotometry and colorimetry were analyzed using UV-visible absorbance spectra and converted to CIELAB coordinates using ColorBySpectra software. Cy mono-acylated on glycosyl position C6’’ had greater spectral absorbance than Cy mono-acylated on glycosyl position C2’’ in pH 4 and pH 6, whereas the di-acylated Cy with acylation on both position C6’’ and C2’’ had greater absorbance than the two mono-acylated regioisomers in pH 4 and pH 6. In pH 2 and 8, negligible differences were observed in 𝜆 vis-max, absorbance at 𝜆 vis-max, as well as their hue and chroma. Cis-acylated Cy had greater spectral and colorimetric differences from their trans-counterpart for monoacylated pigments rather than diacylated pigments. Cis-monoacylated Cy had a shorter half life and greater degradation rate than its trans- isomer, while both isomers of the diacylated Cy degraded at comparable rates, though the trans-diacylated Cy was slightly more stable. Mixture of the monoacylated isomers exhibited behavior that lied between the two isomers, but the mixture of diacylated isomers did not; which suggested that isomeric configuration could make less of an impact on chemical characteristics when greater number of acylations are involved. This dissertation shows the ways in which scientists can manipulate photochromism of ACN to produce colorants with varying chemical characteristics—including color expression and stability in various pH matrices.