Model Studies on the Photosensitized Isomerization of Bixin MARIANA A. MONTENEGRO, ² ALESSANDRO DE O. RIOS, ‡ ADRIANA Z. MERCADANTE, ‡ MO Ä NICA A. NAZARENO, ² AND CLAUDIO D. BORSARELLI* ,² Instituto de Ciencias Quı ´micas, Universidad Nacional de Santiago del Estero, Av. Belgrano (S) 1912, 4200 Santiago del Estero, Argentina, and Departamento de Cie ˆncia de Alimentos, Facultade de Engenharia de Alimentos, UNICAMP, Cx. Postal 6121, 13083-970 Campinas, SP, Brazil The photosensitized isomerization reaction of the natural cis carotenoid bixin (methyl hydrogen 9′- cis-6, 6′-diapocarotene-6, 6′-dioate) with rose bengal or methylene blue as the sensitizer in acetonitrile/ methanol (1:1) solution was studied using UV-vis spectroscopy, high-performance liquid chroma- tography (HPLC), and time-resolved spectroscopic techniques, such as laser-flash photolysis and singlet oxygen phosphorescence detection. In both N 2 - and air-saturated solutions, the main product formed was all-trans-bixin. The observed isomerization rate constants, k obs , decreased in the presence of air or with increase in the bixin concentration, suggesting the participation of the excited triplet state of bixin, 3 Bix*, as precursor of the cisf trans process. On the other hand, bixin solutions in the absence of sensitizer and/or light did not degrade, indicating that the ground state of bixin is stable to thermal isomerization at room temperature. Time-resolved spectroscopic experiments confirmed the formation of the excited triplet state of bixin and its deactivation by ground state bixin and molecular oxygen quenching processes. The primary isomerization products only degraded in the presence of air and under prolonged illumination conditions, probably due to the formation of oxidation products by reaction with singlet molecular oxygen. An energy-transfer mechanism was used to explain the observed results for the bixin transformations, and the consequences for food color are discussed. KEYWORDS: Bixin; photoisomerization; photosensitization; singlet oxygen and triplet states INTRODUCTION Bixin (methyl hydrogen 9′-cis-6, 6′-diapocarotene-6, 6′-dioate, Scheme 1) is the main carotenoid existing in the seed coat of the annatto tree (Bixa orellana L.), which is found in tropical regions of South and Central America, Africa, and Asia (1, 2). Annatto extracts are widely used in the food industry as natural coloring agents in cheese, ice cream, yoghurt, sausages, margarine, snacks, and dressings, and compared to natural colors, are relatively inexpensive, thus increasing their com- mercial relevance (1, 2). The characterization of the coloring components of the annatto extracts (3-5), annatto seeds (6- 9), and their degrada- tion products (10, 11) has received much attention. In particular, the stability of bixin was evaluated under the effect of several factors, such as light, oxygen concentration, temperature, and solvent (12, 13). Bixin showed great stability in the dark, in the presence or absence of oxygen, but under direct illumination was degraded (12). The thermo- and photostability of the 9′- cis double bond in methyl bixin was reported as being so great that the molecule underwent a second transfcis isomerization giving considerable amounts of the 9, 9′-di-cis isomer, but in the presence of iodine and light, 9′-cisftrans isomerization occurred (14). Therefore, it seems that the fade of bixin could be strongly dependent on the experimental conditions (e.g., direct illumination, heating, photosensitization, etc). Most studies on bixin stability focused on thermal degradation (10, 11), direct illumination (12), and the water activity solvent effect (13). In the thermal degradation of bixin, the main products formed were all-trans-bixin, a C17 carotenoid (trans- monomethyl ester of 4,8-dimethyl tetradecahexaenedioic acid) and volatile compounds, especially xylene (5, 10). However, to our knowledge, there is little or no information about the photosensitized degradation of bixin either in model systems or in foods. Moreover, light can dramatically reduce food stability due to the promotion of autoxidation and photosensitized oxidation, the latter being more common, because many natural sensitizers are commonly found in foods. * To whom correspondence should be addressed. Fax: 54-385-4509585. E-mail: cborsa@unse.edu.ar. ² Universidad Nacional de Santiago del Estero. ‡ UNICAMP. Scheme 1. Chemical Structure of the Natural cis-Carotenoid Bixin J. Agric. Food Chem. 2004, 52, 367-373 367 10.1021/jf0349026 CCC: $27.50 © 2004 American Chemical Society Published on Web 12/20/2003